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Substrate assisted catalysis - Application to G proteins
Abstract
The idea that both the substrate and the enzyme contribute to catalysis (substrate assisted catalysis; SAC) is applicable to guanine nucleotide-binding proteins (G proteins). Naturally occurring SAC uses GTP as a general base in the GTPase reaction catalyzed by G proteins. Engineered SAC has identified a putative rate-limiting step for the GTPase reaction and shown that GTPase-deficient oncogenic Ras mutants are not irreversibly impaired. Thus, anti-cancer drugs could potentially be designed to restore the blocked GTPase reaction.
... A straightforward experimental approach to test this hypothesis would be to perform the reaction with an alternate substrate in which the carboxylate of anthranilic acid is replaced by a non-reactive group, for example the amide (Kosloff and Selinger, 2001). However, our efforts to enzymatically synthesize phosphoribosyl-anthranilic amide from anthranilic amide and PRPP by anthranilate phosphoribosyl transferase failed. ...
... Up to now the phenomenon of substrate-assisted catalysis has been discovered in naturally occurring enzymes like GTPases, type II restriction endonucleases or aminoacyl-tRNA synthetases (Dall'Acqua and Carter, 2000;Hussain et al., 2010;Kosloff and Selinger, 2001;So et al., 2011), as well as in engineered serin proteases or GTPases (Dall'Acqua and Carter, 2000;Kosloff and Selinger, 2001). In extension of these hydrolytic reactions, we have shown here that engineered sugar isomerization is presumably facilitated by the participation of the carboxylic acid group of the substrate. ...
... Up to now the phenomenon of substrate-assisted catalysis has been discovered in naturally occurring enzymes like GTPases, type II restriction endonucleases or aminoacyl-tRNA synthetases (Dall'Acqua and Carter, 2000;Hussain et al., 2010;Kosloff and Selinger, 2001;So et al., 2011), as well as in engineered serin proteases or GTPases (Dall'Acqua and Carter, 2000;Kosloff and Selinger, 2001). In extension of these hydrolytic reactions, we have shown here that engineered sugar isomerization is presumably facilitated by the participation of the carboxylic acid group of the substrate. ...
In the course of tryptophan biosynthesis, the isomerization of phosphoribosylanthranilate (PRA) is catalyzed by the (βα)8-barrel enzyme TrpF. The reaction occurs via a general acid–base mechanism with an aspartate and a cysteine residue acting
as acid and base, respectively. PRA isomerase activity could be established on two (βα)8-barrel enzymes involved in histidine biosynthesis, namely HisA and HisF, and on a HisAF chimera, by introducing two aspartate-to-valine
substitutions. We have analyzed the reaction mechanism underlying this engineered activity by measuring its pH dependence,
solving the crystal structure of a HisF variant with bound product analogue, and applying molecular dynamics simulations and
mixed quantum and molecular mechanics calculations. The results suggest that PRA is anchored by the C-terminal phosphate-binding
sites of HisA, HisF and HisAF. As a consequence, a conserved aspartate residue, which is equivalent to Cys7 from TrpF, is
properly positioned to act as catalytic base. However, no obvious catalytic acid corresponding to Asp126 from TrpF could be
identified in the three proteins. Instead, this role appears to be carried out by the carboxylate group of the anthranilate
moiety of PRA. Thus, the engineered PRA isomerization activity is based on a reaction mechanism including substrate-assisted
catalysis and thus differs substantially from the naturally evolved reaction mechanism used by TrpF.
... The mechanism of hydrolysis of guanosine triphosphate (GTP) by G-proteins, leading to guanosine diphosphate (GDP) and inorganic phosphate (Pi), which constitutes one of the most important enzymatic reactions responsible for normal and tumorigenic cellular signal transduction, continues to remain a subject of active debates. [1][2][3][4][5][6][7][8][9][10][11] The most recent publications by Wittinghofer, 11 Li and Zhang, 10 and Pasqualato and Cherfils 12 comprehensively review the subject summarizing advances from structural, kinetic, spectroscopic, and theoretical studies. Two types of the reaction mechanism are usually compared as differentiated by the nature of a transition state. ...
... 13 Another popular model suggests that the substrate GTP itself, specifically g-phosphate group of GTP, serves as the general base in its own hydrolysis. 9 Modeling the catalytic mechanism of the GTP hydrolysis by Ras and RasÁGAP in the complete enzyme has been performed by Warshel and coworkers [14][15][16][17][18][19][20] by using the empirical valence bond (EVB) methodology. 21 In this approach, the diagonal elements of the EVB Hamiltonian (the diabatic energies) describe the energies of valence bond electronic structures, while the different resonant structures are mixed by off-diagonal elements. ...
The hydrolysis reaction of guanosine triphosphate (GTP) by p21(ras) (Ras) has been modeled by using the ab initio type quantum mechanical-molecular mechanical simulations. Initial geometry configurations have been prompted by atomic coordinates of the crystal structure (PDBID: 1QRA) corresponding to the prehydrolysis state of Ras in complex with GTP. Multiple searches of minimum energy geometry configurations consistent with the hydrogen bond networks have been performed, resulting in a series of stationary points on the potential energy surface for reaction intermediates and transition states. It is shown that the minimum energy reaction path is consistent with an assumption of a two-step mechanism of GTP hydrolysis. At the first stage, a unified action of the nearest residues of Ras and the nearest water molecules results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low barrier (16.7 kcal/mol) transition state TS1. At the second stage, the inorganic phosphate is formed in consequence of proton transfers mediated by two water molecules and assisted by the Gln61 residue from Ras. The highest transition state at this segment, TS3, is estimated to have an energy 7.5 kcal/mol above the enzyme-substrate complex. The results of simulations are compared to the previous findings for the GTP hydrolysis in the Ras-GAP (p21(ras)-p120(GAP)) protein complex. Conclusions of the modeling lead to a better understanding of the anticatalytic effect of cancer causing mutation of Gln61 from Ras, which has been debated in recent years.
... A role for Q61 as a general base for activation of the catalytic water is unlikely, but it is thought to be a critical residue in positioning the water molecule during the reaction (Maegley et al., 1996). The role of the Arg finger, inserted into the active site, is to stabilize the negative charge that develops on the bridging oxygen between the β and γ phosphate atoms of the nucleotide during catalysis (Kosloff and Selinger, 2001;Li and Zhang, 2004). In the RasGAP catalyzed reaction, Y32 is in an open conformation, interacting intimately at the Ras/RasGAP interface where it is not involved directly in catalysis. ...
... There is evidence in the literature that the base that activates the water molecule for nucleophilic attack in the hydrolysis reaction is the γ-phosphate of GTP itself, in a substrate assisted catalytic mechanism where the abstracted proton ends up being shared by the P i and the β-phosphate of the GDP leaving group in the product (Kosloff and Selinger, 2001;Pasqualato and Cherfils, 2005). One objection to this mechanism is that electron density would be stabilized at the γphosphorous atom, which is inconsistent with a reaction mechanism where the outcome is an increase in negative charge at the oxygen bridging the β and γ-phosphorus atoms (Maegley et al., 1996). ...
Transformation efficiencies of Ras mutants at residue 61 range over three orders of magnitude, but the in vitro GTPase activity decreases 10-fold for all mutants. We show that Raf impairs the GTPase activity of RasQ61L, suggesting that the Ras/Raf complex differentially modulates transformation. Our crystal structures show that, in transforming mutants, switch II takes part in a network of hydrophobic interactions burying the nucleotide and precatalytic water molecule. Our results suggest that Y32 and a water molecule bridging it to the gamma-phosphate in the wild-type structure play a role in GTP hydrolysis in lieu of the Arg finger in the absence of GAP. The bridging water molecule is absent in the transforming mutants, contributing to the burying of the nucleotide. We propose a mechanism for intrinsic hydrolysis in Raf-bound Ras and elucidate structural features in the Q61 mutants that correlate with their potency to transform cells.
... In some cases when conformational changes are necessary for efficient and sustained activity, these structural rearrangements may provide an activation barrier, that becomes rate limiting 45 . Indeed, as mentioned above, this appears to be the case with Ohr, since the opening and closing of the Arg-loop appears to be crucial for catalysis. ...
Bacteria contain a large repertoire of enzymes to decompose oxidants, such as hydroperoxides. Among them, Organic hydroperoxide resistance (Ohr) proteins play central roles in the bacterial response to fatty acid peroxides and peroxynitrite and present distinct structural and biochemical features in comparison with mammalian Cys-based peroxidases. The molecular events associated with the high reactivity of Ohr enzymes towards hydroperoxides and its reducibility by lipoylated proteins (or dihydrolipoamide) are still elusive. Here, we report six crystallographic structures of two Ohr paralogs from Chromobacterium violaceum, including the complex with dihydrolipoamide. Comparison of these six structures with the other few Ohr structures available in public databases revealed conserved features in the active site, such as a hydrophobic collar. Together with classical, hybrid quantum-classical molecular dynamics simulations and point mutation analyses, we show that Ohr undergoes several structural switches to allow an energetically accessible movement of the loop containing the catalytic Arg, which is stabilized in the closed state when the catalytic Cys is reduced. The structure of Ohr in complex with its substrate (dihydrolipoamide) together with molecular simulations allowed us to characterize the reductive half of the catalytic pathway in detail. Notably, dihydrolipoamide favors Arg-loop closure, thereby assisting enzyme turnover. The conserved physicochemical properties of the Ohr active site and the mechanisms revealed here provide relevant information for the identification of inhibitors with therapeutic potential.
... KRAS P34R shows preservation of intrinsic GTP hydrolysis but a severe defect in GAPmediated hydrolysis . The mechanisms of KRAS Intrinsic and GAPmediated GTP hydrolysis require the receipt of a proton from a catalytic water molecule (Buhrman et al., 2010;Kosloff & Selinger, 2001). In this reaction, a proton is shuttled from the catalytic water molecule via the γ-phosphate of GTP to a nearby bridging water molecule, which can donate hydrogen bonds to both Tyr32 and Gln61 (Figure 3a). ...
RAS proteins are commonly mutated in cancerous tumors, but germline RAS mutations are also found in RASopathy syndromes such as Noonan syndrome (NS) and cardiofaciocutaneous (CFC) syndrome. Activating RAS mutations can be subclassified based on their activating mechanisms. Understanding the structural basis for these mechanisms may provide clues for how to manage associated health conditions. We determined high-resolution X-ray structures of the RASopathy mutant KRASP34R seen in NS and CFCS. GTP and GDP-bound KRASP34R crystallized in multiple forms, with each lattice consisting of multiple protein conformations. In all GTP-bound conformations, the switch regions are not compatible with GAP binding, suggesting a structural mechanism for the GAP insensitivity of this RAS mutant. However, GTP-bound conformations are compatible with intrinsic nucleotide hydrolysis, including one that places R34 in a position analogous to the GAP arginine finger or intrinsic arginine finger found in heterotrimeric G proteins, which may support intrinsic GTP hydrolysis. We also note that the affinity between KRASP34R and RAF-RBD is decreased, suggesting another possible mechanism for dampening of RAS signaling. These results may provide a foothold for development of new mutation-specific strategies to address KRASP34R -driven diseases.
... One of the hypotheses employs the idea of the substrateassisted catalysis. 38 In this mechanism, the terminal functional group of GTP facilitates cleavage of the O-H bond in the nucleophile water molecule synchronized with cleavage of the O 3B -P G bond in GTP (Scheme 2). ...
Mechanism of the deceptively simple reaction of guanosine triphosphate (GTP) hydrolysis catalyzed by the cellular protein Ras in complex with the activating protein GAP is an important issue because of the significance of this reaction in cancer research. We show that molecular modeling of GTP hydrolysis in the Ras-GAP active site reveals a diversity of mechanisms of the intrinsic chemical reaction depending on molecular groups at position 61 in Ras occupied by glutamine in the wild-type enzyme. First, comparison of reaction energy profiles computed at the quantum mechanics/molecular mechanics (QM/MM) level shows that an assignment of the Gln61 side chain in the wild-type Ras either to QM or to MM parts leads to different scenarios corresponding to the glutamine-assisted or the substrate-assisted mechanisms. Second, replacement of Gln61 by the nitro-analog of glutamine (NGln) or by Glu, applied in experimental studies, results in two more scenarios featuring the so-called two-water and the concerted-type mechanisms. The glutamine-assisted mechanism in the wild-type Ras-GAP, in which the conserved Gln61 plays a decisive role, switching between the amide and imide tautomer forms, is consistent with the known experimental results of structural, kinetic and spectroscopy studies. The results emphasize the role of the Ras residue Gln61 in Ras-GAP catalysis and explain the retained catalytic activity of the Ras-GAP complex towards GTP hydrolysis in the Gln61NGln and Gln61Glu mutants of Ras.
... The fine details of the mechanisms of GTP hydrolysis by this enzyme have been the subject of substantial debate, focusing on not just the nature of the transition state (associative vs. dissociative), 22 but also the feasibility of substrate-vs. general-base assisted catalysis, [34][35][36]92,[153][154][155][156][157] as well as the involvement of one vs. two water molecules at the transition state for GTP hydrolysis. ...
Phosphate ester hydrolysis is fundamental to many life processes, and has been the topic of substantial experimental and computational research effort. However, even the simplest of phosphate esters can be hydrolyzed through multiple possible pathways that can be difficult to distinguish between, either experimentally, or computationally. Therefore, the mechanisms of both the enzymatic and non-enzymatic reactions have been historically controversial. In the present contribution, we highlight a number of technical issues involved in reliably modeling these computationally challenging reactions, as well as proposing potential solutions. We also showcase examples of our own work in this area, discussing both the non-enzymatic reaction in aqueous solution, as well insights obtained from the computational modeling of organophosphate hydrolysis and catalytic promiscuity amongst enzymes that catalyze phosphoryl transfer.
... The distinction between binding and catalysis discussed above for enzyme functional groups is valid also for groups carried by the substrate. The concept of substrate assisted catalysis, where a substrate functional group participates directly in catalysis to increase k cat , is a topic much discussed in literature [51][52][53]. ...
... Switch II accommodates the J phosphate of GTP and is necessary for the GTP hydrolysis reaction (Sprang, 1997;Kosloff & Selinger, 2001). This structural element often undergoes large rearrangements when GTP replaces GDP on the GTPase (Berchtold et al., 1993;Kjeldgaard et al., 1993;Sprang, 1997;Nissen et al., 1999). ...
... 13,14 Gln61 is essential for GTP hydrolysis, 70 although its exact role has been a source of intense debate for more than a decade. 13,14,16,67,73,74 A detailed discussion of its possible role can be found in Shurki and Warshel,14 where the authors show that Gln61 is coupled to other residues in the P-loop, switch I, 3, and switch II regions and mutations of Gln 61 lead to a major disturbance in the preorganized environment. Here we just report on the destabilization suffered by this residue upon binding to GAP, which would confirm previous results on its implication in catalysis. ...
Finding why protein–protein interactions (PPIs) are so specific can provide a valuable tool in a variety of fields. Complexes are frequently clustered according to their lifetime (transient or
permanent), their composition (homodimeric or heterodimeric) and even the posibility (or not) of finding the unbound partners alone in vivo (non-obligate and obligate). Although this classification is arbitrary [1], a common feature for strong complexes is a somewhat more hydrophobic character in the interacting region than in the rest of the molecular surface. For soft interactions, on the contrary, it is difficult to find a similar correlation. Thus, it appears that, in general, transient complexes tend to involve the interaction between two regions at least equally polar than the rest of the surface. PPIs have to compete with both the interaction between surface residues with water and the interaction between surface residues themselves in the unbound proteins. On the other hand, several works by Warshel and others have shown that the notion of active site electrostatic preorganization can be used to interpret the high efficiency in enzyme reactions (see, e.g. [2] for a review). It has been shown that this pre-organization can be related to the stability of the residues in this region. In some enzymes, in addition, conformational changes upon binding to other proteins lead to an increase in the activity of the enzymatic partner. These facts suggest that the evaluation of the stability of residues in a protein can be used to detect active site regions and eventually to assign functionality to orphan proteins. Following these arguments, we will try to extend the pre-organization theory to PPIs.
... In most proteins where nucleotides regulate biological activity, NTP and NDP ‡ stabilize alternate conformations of the protein and catalytic turnover at the site is extremely slow. For instance, G-proteins involved in signal transduction pathways are activated by GTP but are inactive with GDP bound; slow hydrolysis of the bound GTP (k cat ~ 10 −2 -10 −3 min −1 ) acts as a "switch mechanism" that allows regulation of biological activity (35). Within this context, ATP hydrolysis by the assembly site in the terminase mix is unusual. ...
Terminase enzymes are responsible for the excision of a single genome from a concatemeric precursor (genome maturation) and concomitant packaging of DNA into the capsid shell. Here, we demonstrate that lambda terminase can be purified as a homogeneous "protomer" species, and we present a kinetic analysis of the genome maturation and packaging activities of the protomeric enzyme. The protomer assembles into a distinct maturation complex at the cos sequence of a concatemer. This complex rapidly nicks the duplex to form the mature left end of the viral genome, which is followed by procapsid binding, activation of the packaging ATPase, and translocation of the duplex into the capsid interior by the terminase motor complex. Genome packaging by the protomer shows high fidelity with only the mature left end of the duplex inserted into the capsid shell. In sum, the data show that the terminase protomer exhibits catalytic activity commensurate with that expected of a bone fide genome maturation and packaging complex in vivo and that both catalytically competent complexes are composed of four terminase protomers assembled into a ringlike structure that encircles duplex DNA. This work provides mechanistic insight into the coordinated catalytic activities of terminase enzymes in virus assembly that can be generalized to all of the double-stranded DNA viruses.
... Another approach explored for attenuating Ras signaling consists of the use of molecules that promote the GTP hydrolysis of oncogenic Ras mutants (Ras inactivation; Figure 2, Scheme 3b), such as 3,4-diaminobenzophenone-phosphonoamidate of GTP (DABP-GTP). This GTP analog is able to rescue the defective GTPase reaction of the oncogenic Ras version carrying a mutation in residue Gln61, since its aromatic amino group substitute the function of the carbonamide side chain of Gln6 in promoting GTP hydrolysis reaction [122,123]. Since DABP-GTP show problems of specificity and delivery, it requires optimization for the clinical development. ...
Introduction:
Ras proteins are small GTPases molecular switches that cycle through two alternative conformational states, a GDP-bound inactive state and a GTP-bound active state. In the active state, Ras proteins interact with and modulate the activity of several downstream effectors regulating key cellular processes including proliferation, differentiation, survival, senescence, migration and metabolism. Activating mutations of RAS genes and of genes encoding Ras signaling members have a great incidence in proliferative disorders, such as cancer, immune and inflammatory diseases and developmental syndromes. Therefore, Ras and Ras signaling represent important clinical targets for the design and development of pharmaceutically active agents, including anticancer agents.
Areas covered:
The authors summarize methods available to down-regulate the Ras pathway and review recent patents covering Ras signaling modulators, as well as methods designed to kill specifically cancer cells bearing activated RAS oncogene.
Expert opinion:
Targeted therapy approach based on direct targeting of molecules specifically altered in Ras-dependent diseases is pursued with molecules that down-regulate expression or inhibit the biological function of mutant Ras or Ras signaling members. The low success rate in a clinical setting of molecules targeting activated members of the Ras pathway may require development of novel approaches, including combined and synthetic lethal therapies.
... 22 Thus, it was proposed that in the catalytic mechanism of HisRS O pro-S acted as a general base and deprotonated the hydroxyl of the A76 residue of the tRNA cosubstrate as shown in Scheme 1. 16 Based on their observed dissimilar active sites and apparent common utilization of a non-bridging phospho-oxygen of the substrate as the mechanistic base, it has been suggested that for aaRS's such a substrate-assisted catalysis (SAC) mechanism may be a general feature of these presumably ancient enzymes. 5,16,[22][23][24][25][26] Following these experimental studies we performed a detailed density functional theory (DFT)-based computational investigation on the catalytic mechanism of HisRS. 5 In particular, the ability of the aa-AMP's bridging and pro-R and pro-S non-bridging oxygens to act as a base and the mechanism by which they may catalyse the aminoacylation reaction was systematically examined. ...
Density functional theory-based methods in combination with large chemical models have been used to investigate the mechanism of the second half-reaction catalyzed by Thr-tRNA synthetase: aminoacyl transfer from Thr-AMP onto the (A76)3'OH of the cognate tRNA. In particular, we have examined pathways in which an active site His309 residue is either protonated or neutral (i.e., potentially able to act as a base). In the protonated His309-assisted mechanism, the rate-limiting step is formation of the tetrahedral intermediate. The barrier for this step is 155.0 kJ mol(-1), and thus, such a pathway is concluded to not be enzymatically feasible. For the neutral His309-assisted mechanism, two models were used with the difference being whether Lys465 was included. For either model, the barrier of the rate-limiting step is below the upper thermodynamic enzymatic limit of ~125 kJ mol(-1). Specifically, without Lys465, the rate-limiting barrier is 122.1 kJ mol(-1) and corresponds to a rotation about the tetrahedral intermediate C(carb)-OH bond. For the model with Lys465, the rate-limiting barrier is slightly lower and corresponds to the formation of the tetrahedral intermediate. Importantly, for both "neutral His309" models, the neutral amino group of the threonyl substrate directly acts as the proton acceptor; in the formation of the tetrahedral intermediate, the (A76)3'OH proton is directly transferred onto the Thr-NH(2). Therefore, the overall mechanism follows a general substrate-assisted catalytic mechanism.
... It is now established that intrinsic hydrolysis in Ras occurs through a loose, dissociative-like transition state (TS) with significant concerted character, as does the GAP-catalyzed reaction (26). This does not rule out a mechanism in which the GTP itself receives a proton from the catalytic water molecule (27). Within these parameters, we propose a general outline for catalysis deduced from our structure of the ground state of Ras with an activated allosteric switch, augmenting the mechanism we previously published (17). ...
Ras and its effector Raf are key mediators of the Ras/Raf/MEK/ERK signal transduction pathway. Mutants of residue Q61 impair the GTPase activity of Ras and are found prominently in human cancers. Yet the mechanism through which Q61 contributes to catalysis has been elusive. It is thought to position the catalytic water molecule for nucleophilic attack on the gamma-phosphate of GTP. However, we previously solved the structure of Ras from crystals with symmetry of the space group R32 in which switch II is disordered and found that the catalytic water molecule is present. Here we present a structure of wild-type Ras with calcium acetate from the crystallization mother liquor bound at a site remote from the active site and likely near the membrane. This results in a shift in helix 3/loop 7 and a network of H-bonding interactions that propagates across the molecule, culminating in the ordering of switch II and placement of Q61 in the active site in a previously unobserved conformation. This structure suggests a direct catalytic role for Q61 where it interacts with a water molecule that bridges one of the gamma-phosphate oxygen atoms to the hydroxyl group of Y32 to stabilize the transition state of the hydrolysis reaction. We propose that Raf together with the binding of Ca(2+) and a negatively charged group mimicked in our structure by the acetate molecule induces the ordering of switch I and switch II to complete the active site of Ras.
... It should be noted that none of the available experiments can determine whether the mechanism is associative or dissociative [32]. Another popular model suggests that the substrate GTP itself, specifically, γ-phosphate group of GTP, serves as the general base in its own hydrolysis [33,34]. ...
Elongation factor Tu (EF-Tu), the protein responsible for delivering aminoacyl-tRNAs (aa-tRNAs) to ribosomal A site during translation, belongs to the group of guanosine-nucleotide (GTP/GDP) binding proteins. Its active 'on'-state corresponds to the GTP-bound form, while the inactive 'off'-state corresponds to the GDP-bound form. In this work we focus on the chemical step, GTP+H(2)O-->GDP+Pi, of the hydrolysis mechanism. We apply molecular modeling tools including molecular dynamics simulations and the combined quantum mechanical-molecular mechanical calculations for estimates of reaction energy profiles for two possible arrangements of switch II regions of EF-Tu. In the first case we presumably mimic binding of the ternary complex EF-Tu.GTP.aa-tRNA to the ribosome and allow the histidine (His85) side chain of the protein to approach the reaction active site. In the second case, corresponding to the GTP hydrolysis by EF-Tu alone, the side chain of His85 stays away from the active site, and the chemical reaction GTP+H(2)O-->GDP+Pi proceeds without participation of the histidine but through water molecules. In agreement with the experimental observations which distinguish rate constants for the fast chemical reaction in EF-Tu.GTP.aa-tRNA.ribosome and the slow spontaneous GTP hydrolysis in EF-Tu, we show that the activation energy barrier for the first scenario is considerably lower compared to that of the second case.
... Involvement of substrate substituents in promoting substrate reactivity on enzymes [substrate-assisted catalysis (1)(2)(3)(4)(5)] and enzyme residues in perturbing substrate geometry [substrate distortion (6)(7)(8)(9)(10)] are among recognized factors that can enhance rates of enzyme catalysis. Here these influences are investigated with respect to the catalytic mechanism of scytalone dehydratase. ...
Alternative substrates and site-directed mutations of active-site residues are used to probe factors controlling the catalytic efficacy of scytalone dehydratase. In the E1cb-like, syn-elimination reactions catalyzed, efficient catalysis requires distortion of the substrate ring system to facilitate proton abstraction from its C2 methylene and elimination of its C3 hydroxyl group. Theoretical calculations indicate that such distortions are more readily achieved in the substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) than in the physiological substrates vermelone and scytalone by approximately 2 kcal/mol. A survey of 12 active-site amino acid residues reveals 4 site-directed mutants (H110N, N131A, F53A, and F53L) have higher relative values of k(cat) and k(cat)/K(m) for DDBO over scytalone and for DDBO over vermelone than the wild-type enzyme, thus suggesting substrate-distortion roles for the native residues in catalysis. A structural link for this function is in the modeled enzyme-substrate complex where F53 and H110 are positioned above and below the substrate's C3 hydroxyl group, respectively, for pushing and pulling the leaving group into the axial orientation of a pseudo-boat conformation; N131 hydrogen-bonds to the C8 hydroxyl group at the opposite end of the substrate, serving as a pivot for the actions of F53 and H110. Deshydroxyvermelone lacks the phenolic hydroxyl group and the intramolecular hydrogen bond of vermelone. The relative values of k(cat) (95) and k(cat)/K(m) (1800) for vermelone over deshydroxyvermelone for the wild-type enzyme indicate the importance of the hydroxyl group for substrate recognition and catalysis. Off the enzyme, the much slower rates for the solvolytic dehydration of deshydroxyvermelone and vermelone are similar, thus specifying the importance of the hydroxyl group of vermelone for enzyme catalysis.
... These results show for the first time that the GTPase switch of oncogenic Ras proteins is not irreversibly damaged. DABP-GTP is a substrate for GTPases and it simultaneously contributes to catalysis via its' exocyclic amine group, a phenomenon which is called substrate-assisted catalysis (Kosloff & Selinger, 2001). Obviously GTP analogues themselves, such as DABP-GTP, are not good lead compounds, as the high GTP affinity for Ras and the high concentration of GTP in the cells renders this approach unfeasible. ...
... The basic mechanism of GTPase stimulation relies on the stabilization of the highly mobile switch regions and the transition state of the GTP-hydrolysis reaction by supplying a catalytic arginine to the active site (Gamblin & Smerdon, 1998;Scheffzek et al, 1998;Kosloff & Selinger, 2001;Vetter & Wittinghofer, 2001). Therefore, GAPs position the catalytically crucial Gln63 in an appropriate conformation towards a nucleophilic water molecule, which hydrolyses GTP and neutralizes developing negative charges on the leaving group during the phosphoryl-transfer reaction. ...
The signalling functions of Rho-family GTPases are based on the formation of distinctive protein-protein complexes. Invaluable insights into the structure-function relationships of the Rho GTPases have been obtained through the resolution of several of their structures in complex with regulators and downstream effectors. In this review, we use these complexes to compare the binding and specificity-determining sites of the Rho GTPases. Although the properties that characterize these sites are diverse, some fundamental conserved principles that govern their intermolecular interactions have emerged. Notably, all of the interacting partners of the Rho GTPases, irrespective of their function, bind to a common set of conserved amino acids that are clustered on the surface of the switch regions. This conserved region and its specific structural characteristics exemplify the convergence of the Rho GTPases on a consensus binding site.
... Because neither hydroxyl is chemically transformed during the step that it promotes, we concluded that Rnl2 employs a mechanism of substrate-assisted catalysis. Substrate-assisted catalysis has been invoked for a variety of natural and engineered enzymes, including serine proteases, GTPases, acylphosphatases, DNA glycosylases, and the ribosome (22)(23)(24)(25)(26). ...
T4 RNA ligase 2 (Rnl2) efficiently seals 3′-OH/5′-PO4RNA nicks via three nucleotidyl transfer steps. Here we show that the terminal 3′-OH at the nick accelerates the second step
of the ligase pathway (adenylylation of the 5′-PO4 strand) by a factor of 1000, even though the 3′-OH is not chemically transformed during the reaction. Also, the terminal
2′-OH at the nick accelerates the third step (attack of the 3′-OH on the 5′-adenylated strand to form a phosphodiester) by
a factor of 25–35, even though the 2′-OH is not chemically reactive. His-37 of Rnl2 is uniquely required for step 3, providing
a ∼102 rate acceleration. Biochemical epistasis experiments show that His-37 and the RNA 2′-OH act independently. We conclude that
the broken RNA end promotes catalysis of its own repair by Rnl2 via two mechanisms, one of which (enhancement of step 3 by
the 2′-OH) is specific to RNA ligation. Substrate-assisted catalysis provides a potential biochemical checkpoint during nucleic
acid repair.
... 13,14 Gln61 is essential for GTP hydrolysis, 70 although its exact role has been a source of intense debate for more than a decade. 13,14,16,67,73,74 A detailed discussion of its possible role can be found in Shurki and Warshel,14 where the authors show that Gln61 is coupled to other residues in the P-loop, switch I, 3, and switch II regions and mutations of Gln 61 lead to a major disturbance in the preorganized environment. Here we just report on the destabilization suffered by this residue upon binding to GAP, which would confirm previous results on its implication in catalysis. ...
Finding why protein-protein interactions (PPIs) are so specific can provide a valuable tool in a variety of fields. Statistical surveys of so-called transient complexes (like those relevant for signal transduction mechanisms) have shown a tendency of polar residues to participate in the interaction region. Following this scheme, residues in the unbound partners have to compete between interacting with water or interacting with other residues of the protein. On the other hand, several works have shown that the notion of active site electrostatic preorganization can be used to interpret the high efficiency in enzyme reactions. This preorganization can be related to the instability of the residues important for catalysis. In some enzymes, in addition, conformational changes upon binding to other proteins lead to an increase in the activity of the enzymatic partner. In this article the linear response approximation version of the semimacroscopic protein dipoles Langevin dipoles (PDLD/S-LRA) model is used to evaluate the stability of several residues in two phosphate hydrolysis enzymes upon complexation with their activating partners. In particular, the residues relevant for PPI and for phosphate hydrolysis in the CDK2/Cyclin A and Ras/GAP complexes are analyzed. We find that the evaluation of the stability of residues in these systems can be used to identify not only active site regions but it can also be used as a guide to locate "hot spots" for PPIs. We also show that conformational changes play a major role in positioning interfacing residues in a proper "energetic" orientation, ready to interact with the residues in the partner protein surface. Thus, we extend the preorganization theory to PPIs, extrapolating the results we obtained from the above-mentioned complexes to a more general case. We conclude that the correlation between stability of a residue in the surface and the likelihood that it participates in the interaction can be a general fact for transient PPIs.
... The repair of defective GTPase activity of mutant RAS by GTP derivatives bearing residues required for GTP hydrolysis has been reported [167,168]. However, this avenue of RAS inhibition still awaits additional investigation to solve its major problems, such as specificity or transport of compounds modified by triphosphates through biological membranes. ...
RAS proteins are small GTPases, which serve as master regulators of a myriad of signaling cascades involved in highly diverse cellular processes. RAS oncogenes have been originally discovered as retroviral oncogenes, and ever since constitutively activating RAS mutations have been identified in human tumors, they are in the focus of intense research. In this review, we summarize the biochemical properties of RAS proteins, trace down the evolution of RAS signaling and present an overview of the spatio-temporal activation of major RAS isoforms. We further discuss RAS effector pathways, their role in normal and transformed cell physiology and summarize ongoing attempts to interfere with aberrant RAS signaling. Finally, we comment on the role of micro RNAs in modulating RAS expression, contribution of RAS to stem cell function and on high-throughput analyses of RAS signaling networks.
... (3) Substrate-assisted catalysis. Another catalytic strategy that may be operative for R67 DHFR is substrate assisted catalysis (11,55,56). As noted above, the adenosyl-2′-phosphate group of the NADP + could provide a useful proton source for N5 protonation, although no enzyme-facilitated pathway has been identified. ...
Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR.DHF.NADP+ catalytic complex, resolved to 1.26 A. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine ring. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67'-Y69' residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationally challenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation.
Ohrs (organic hydroperoxide resistance proteins) are antioxidant enzymes that play central roles in the response of microorganisms to organic peroxides. Here, we describe recent advances in the structure, catalysis, phylogeny, regulation, and physiological roles of Ohr proteins and of its transcriptional regulator, OhrR, highlighting their unique features. Ohr is extremely efficient in reducing fatty acid peroxides and peroxynitrite, two oxidants relevant in host-pathogen interactions. The highly reactive Cys residue of Ohr, named peroxidatic Cys (Cp), composes together with an arginine and a glutamate the catalytic triad. The catalytic cycle of Ohrs involves a condensation between a sulfenic acid (Cp-SOH) and the thiol of the second conserved Cys, leading to the formation of an intra-subunit disulfide bond, which is then reduced by dihydrolipoamide or lipoylated proteins. A structural switch takes place during catalysis, with the opening and closure of the active site by the so-called Argloop. Ohr is part of the Ohr/OsmC super family that also comprises OsmC and Ohr-like proteins. Members of the Ohr, OsmC and Ohr-like subfamilies present low sequence similarities among themselves, but share a high structural conservation, presenting two Cys residues in their active site. The pattern of gene expression is also distinct among members of the Ohr/OsmC subfamilies. The expression of ohr genes increases upon organic hydroperoxides treatment, whereas the signals for the upregulation of osmC are entry into the stationary phase and/or osmotic stress. For many ohr genes, the upregulation by organic hydroperoxides is mediated by OhrR, a Cys-based transcriptional regulator that only binds to its target DNAs in its reduced state. Since Ohrs and OhrRs are involved in virulence of some microorganisms and are absent in vertebrate and vascular plants, they may represent targets for novel therapeutic approaches based on the disruption of this key bacterial organic peroxide defense system.
IntroductionSignaling Pathways Controlling Cell SurvivalConclusions
The free energy profiles for the chemical reaction of the guanosine triphosphate hydrolysis GTP +H2 O →GDP +Pi by Ras-GAP for the wild-type and G13V mutated Ras were computed by using molecular dynamics protocols with the QM(ab initio)/MM potentials. The results are consistent with the recent measurements of reaction kinetics in Ras-GAP showing about two-order reduction of the rate constant upon G13V mutation in Ras: the computed activation barrier on the free energy profile is increased by 3 kcal/mol upon the G13V replacement. The major reason for a higher energy barrier is a shift of the 'arginine finger' (R789 from GAP) from the favorable position in the active site. The results of simulations provide support for the mechanism of the reference reaction according to which the Q61 side chain directly participates in chemical transformations at the proton transfer stage. This article is protected by copyright. All rights reserved.
© 2015 Wiley Periodicals, Inc.
This chapter discusses the role of purine-binding enzymes as cancer targets. It also provides an overview of kinesin spindle protein (KSP), a member of kinesin superfamily of MT motors, which convert the energy released from ATP hydrolysis into mechanical force for transport along microtubules in the cell. The ideal target for small molecule drug discovery is one that is essential for the disease state, yet nonessential for normal tissues. One recently emerged class of drugable targets is the protein kinases, exemplified by bcr-abl, which is the target of the highly successful new CML drug, Gleevec. The common drugable feature of the kinase class is the ability to bind ATP. The ATP purine moiety is bound in a hydrophobic environment of the active site and stabilized by key hydrogen bonds. Such a site favors binding of flat, aromatic heterocycles, a class of compound that has proven amenable to optimization and drug development, thus, making kinases eminently drugable. However, kinases are not the only class of enzyme that binds purines through key hydrogen bonds and hydrophobic stacking interactions; other such purine-binding enzymes include ATPases, GTPases, sulfotransferases, etc. Thus, these enzymes offer additional possibilities for drug discovery. To illustrate the potential of this emerging class of drug targets, this chapter focuses on the oncology and specifically on ATPases, GTPases and sulfotransferases.
The reaction path for the catalytic conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) by the enzyme mammalian adenylyl cyclase has been calculated theoretically using the Hartree−Fock method. The crystal structure of a thiophosphate reactant analogue, ATPαS, provides the basic structure of the active site binding that is then leveraged into the native reaction path by energy gradient optimization of protein binding residues and the ATP. A two-metal cluster bound to two aspartate residues and the ATP is important both structurally and catalytically. Autocatalytic activation of the reacting ribose 3‘OH group is calculated in the reactant conformation but the catalytic MgA divalent cation binds to the developing 3‘O anion and stabilizes the formation of a five-coordinate intermediate with the cyclic phosphate already formed. Changes in the coordination of the metals in the complex and the H-bonding of arginines that bridge the phosphate groups stabilize the reaction path complex from reactive intermediate to the product. Final transfer of the 3‘H proton to the oxygen bridging the α and β phosphate groups yields the cAMP and pyrophosphate product still bound by many H-bonds in the active site.
Binding and hydrolysis of ampicillin are described in a model active site derived from dinuclear B. fragilis zinc lactamase. The protein binding site consists of the two zinc cations bound with a bridging hydroxide and ligands from the first-shell residues, conserved residues near the zinc site, and the moveable loop of residues from numbers 43−53. The model active site consists of the first-shell residues, the conserved residues, and glu45 and glu47 from the moveable loop. Ampicillin is primarily located in the active site by the binding of the thiazolidine ring's extra-cyclic carboxylate to the ammonium of conserved lysine 184 when water bound to Zn2 in the active site is retained. A comparable strong salt bridge is formed between the ammonium of the ampicillin zwitterion and glu45 on the flexible loop that moves mostly as a unit at least 10 Å to complete the binding site. The zwitterion character of this antibiotic influences the final docking arrangement and ultimate reaction path. Classical molecular dynamics, in the presence of Zn2 bound water, Wat1 and Wat2, and a limited number of waters placed around the ionic groups in the active site, determined a number of reactive docking conformations. One of the low-energy structures with strong interactions to glu45 and glu47 was chosen by the reactive proximity of the nucleophilic water, Wat2, to calculate the reaction path for binding reactant, intermediates, and product for the initial hydrolysis reaction. Water is added to solvate the classical reactant structure, and the reaction path was calculated quantum mechanically within a model chosen from the molecular mechanics structure. Two waters were found in a productive conformation for hydrolysis, the water bound to Zn2 (path 1) and water bound to the ampicillin carboxylate (path 2). In path 1, the hydrolysis product is only bound to the enzyme through hydrogen bonds and can be released by solvating these bonds. Additional proton-transfer steps from the initial product can occur, however, to create intermediates from this product stabilized by interaction with the Zn1 cation. The product formed in path 2 is bound directly to Zn2 suggesting that neither zinc is specially chosen for a catalytic role. Within this model the entire active site is utilized for both binding and catalysis in the case of ampicillin. Strong polar hydrogen bonds are found to the substrate, the waters in the active site, and the residue ligands present in the active site. Autocatalysis or assistance in water activation by the carboxylate of the antibiotic is found and likely to be general. The proton abstracted from the water can park on a number of anionic or polar atom sites in the active site leading to a range of intermediates. The lactam ring C−N bond does not break with prior protonation of the nitrogen or with the initial attack by the hydroxide abstracted from the nucleophilic water but requires attack of the hydroxide at the carbonyl carbon either prior to proton binding or concurrently. This study provides insight into a wider variety of antibiotic docking and shows that more than one reaction path is possible within the highly ionic active site of a bimetallic lactamase.
BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q61K) from shrimp Penaeus japonicus (Huang et al., 2000). The GTPase-activating protein (GAP) in the cytosol fraction was significantly expressed and degraded, compared to untransformed cells on the western blot. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with GAP was investigated using GAP purified from mouse brains. SDS-polyacrylamide gel electrophoresis revealed the monomers of the purified GAP to have a relative mass of 65,000. Since the purified GAP was bound to the Ras conjugated affinity sepharose column with high affinity and its GTP hydolysis activity upon binding with tubulin was suppressed, the purified enzyme was concluded to be neurofibromin-like. The purified GAP enhanced the intrinsic GTPase activity of the S-Ras, to convert it into the inactive GDP-bound form, in agreement with findings for GTP-bound KB-Ras in vitro. To compare the effects between isoprenoids and GAP on the GTP-hydrolysis of Ras, we applied the GTP-locked shrimp mutant S-Ras(Q61K) and GTP-locked rat mutant KB-ras(Q61K). Radioassay studies showed that geranylgeranyl pyrophosphate at μg level catalyzed the GTP hydrolysis of S-Ras(Q61K) and KB-ras(Q61K) competently, but not farnesyl pyrophosphate or the purified GAP. The present study provides the view that the geranylgeranyl pyrophosphate at carboxyl terminal CAAX assists GTP hydrolysis to Ras proteins probably in a manner similar to the substrate assisted catalysis in GTPase mechanism. J. Exp. Zool. 290:642–651, 2001. © 2001 Wiley-Liss, Inc.
Results of simulation of the mechanism of hydrolysis of adenosine triphosphate and guanosine triphosphate in protein matrices,
as well as of deprotonated methyl triphosphate in water clusters by quantum and molecular mechanics with separation of the
reaction system into conformationally flexible effective fragments are discussed.
Activation of the water molecule involved in GTP hydrolysis within the HRas·RasGAP system is analyzed using a tailored approach based on hybrid quantum mechanics/molecular mechanics (QM/MM) simulation. A new path emerges: transfer of a proton from the attacking water molecule to a second water molecule, then a different proton is transferred from this second water molecule to the GTP. Gln(61) will stabilize the transient OH(-) and H(3)O(+) molecules thus generated. This newly proposed mechanism was generated by using, for the first time to our knowledge, the entire HRas-RasGAP protein complex in a QM/MM simulation context. It also offers a rational explanation for previous experimental results regarding the decrease of GTPase rate found in the HRas Q61A mutant and the increase exhibited by the HRas Q61E mutant.
Bayreuth, Universiẗat, Diss., 2002 (Nicht für den Austausch).
Eine der größten Herausforderungen der Medizin ist die Therapie der Volkskrankheit Diabetes mellitus. Die überwiegende Mehrheit der betroffenen Patienten erkrankt an Typ 2-Diabetes, bei dem die Insulin-stimulierte Translokation des Glucosetransporters GLUT4 zur Plasmamembran in Zellen des peripheren Gewebes stark reduziert ist. Für die Regulation des intrazellulären Transports von GLUT4-enthaltenden Membranvesikeln sind Rab-Proteine wie Rab11A von zentraler Bedeutung. Diese kleinen GTP-bindenden Proteine koordinieren aufgrund ihrer interkonvertiblen Konformationen den endo- und exozytotischen Vesikeltransport sowie die Fusion der Vesikel mit der Plasmamembran. Die Ergebnisse der vorliegenden Arbeit aus Untersuchungen der Insulin-stimulierten Glucoseaufnahme und Translokation von GLUT4 zur Plasmamembran sowie der subzellulären Lokalisation von GLUT4-enthaltenden Membranvesikeln in primären humanen Skelettmuskelzellen und in Zellen einer Ratten-Kardiomyoblasten-Zelllinie zeigen, dass Rab11A an der Sequestrierung des Glucosetransporters beteiligt ist und eine Verteilerfunktion am Ausgang des Endosomalen Recycling-Kompartiments in Insulin-sensitiven Muskelzellen besitzt. Die Überexpression der dominant-negativen Mutante Rab11A N124I führte zu einer Reduktion der Insulinwirkung, die aus einer Verschiebung der GLUT4-enthaltenden Membranvesikel in Insulin-insensitive Kompartimente resultiert. Außerdem wird durch den Verlust der GTP-Bindungsstelle bei Rab11A N124I die Assoziation akzessorischer Proteine wie der Rab-spezifischen Regulator- bzw. Effektorproteine inhibiert, die die Wirkung von Rab11A während des Transport der GLUT4-Speichervesikel entlang des Aktin-Zytoskeletts und die Fusion mit der Plasmamembran vermitteln. Die Untersuchungen zum Einfluss von Rab11A auf die Insulin-stimulierte Translokation ergaben, dass die Rab-GTPase in Insulin-sensitiven Muskelzellen als Sortierprotein am Ausgang des Endosomalen Recycling-Kompartiments agiert und somit endozytotische Transportprozesse reguliert. Außerdem kann aufgrund der Wirkung der dominant-negativen Mutante von Rab11A eine Beteiligung der Rab-GTPase an der Regulation des exozytotischen Vesikeltransport postuliert werden.
Purines are critical cofactors in the enzymatic reactions that create and maintain living organisms. In humans, there are approximately 3,266 proteins that utilize purine cofactors and these proteins constitute the so-called purinome. The human purinome encompasses a wide-ranging functional repertoire and many of these proteins are attractive drug targets. For example, it is estimated that 30% of modern drug discovery projects target protein kinases and that modulators of small G-proteins comprise more than 50% of currently marketed drugs. Given the importance of purine-binding proteins to drug discovery, the following review will discuss the forces that mediate protein:purine recognition, the factors that determine druggability of a protein target, and the process of structure-based drug design. A review of purine recognition in representatives of the various purine-binding protein families, as well as the challenges faced in targeting members of the purinome in drug discovery campaigns will also be given.
Density functional theory methods have been used to investigate possible mechanisms of the second half-reaction of aminoacylation catalyzed by histidyl-tRNA synthetase: transfer of the aminoacyl moiety from histidyl-adenylate to the terminal adenosine (A76) of tRNA. The properties of the two mechanistically important nonbridging phosphate oxygens of the histidyl-adenylate in the substrate-bound complex were first considered. It is found that the nonbridging pro-S oxygen is slightly more basic than the pro-R oxygen due to the fact that the former is involved in a weaker hydrogen bonding network than the latter. Three possible mechanisms in which the proton of the 3'-OH group of A76 transfers to the bridging phosphate oxygen and the nonbridging pro-R and -S oxygens were then investigated. When the bridging phosphate oxygen acts as the base, the reaction occurs via a four-membered ring transition structure with a considerably high barrier. When the pro-R oxygen acts as the base, a concerted mechanism was again found. However, it proceeds via a six-membered ring transition structure. In contrast, when the pro-S oxygen acts as a base, an associative stepwise mechanism was found which, furthermore, also had the lowest barrier of the three mechanisms obtained. Comparisons of these three mechanisms and reasons for the differences in barriers are also provided.
ADP-ribosylation controls many processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARP6-PARP16, raising questions about how these enzymes function. We find that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has transferase rather than polymerase activity. Despite this fundamental difference, PARP10 also modifies acidic residues. Consequently, we propose an alternative catalytic mechanism for PARP10 compared to PARP1 in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate by using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells.
Uracil phosphoribosyltransferase (UPRT) is a member of a large family of salvage and biosynthetic enzymes, the phosphoribosyltransferases, and catalyzes the transfer of ribose 5-phosphate from alpha-d-5-phosphoribosyl-1-pyrophosphate (PRPP) to the N1 nitrogen of uracil. The UPRT from the opportunistic pathogen Toxoplasma gondii represents a promising target for rational drug design, because it can create intracellular, lethal nucleotides from subversive substrates. However, the development of such compounds requires a detailed understanding of the catalytic mechanism. Toward this end we determined the crystal structure of the T. gondii UPRT bound to uracil and cPRPP, a nonhydrolyzable PRPP analogue, to 2.5-A resolution. The structure suggests that the catalytic mechanism is substrate-assisted, and a tetramer would be the more active oligomeric form of the enzyme. Subsequent biochemical studies revealed that GTP binding, which has been suggested to play a role in catalysis by other UPRTs, causes a 6-fold activation of the T. gondii enzyme and strikingly stabilizes the tetramer form. The basis for stabilization was revealed in the 2.45-A resolution structure of the UPRT-GTP complex, whereby residues from three subunits contributed to GTP binding. Thus, our studies reveal an allosteric mechanism involving nucleotide stabilization of a more active, higher order oligomer. Such regulation of UPRT could play a role in the balance of purine and pyrimidine nucleotide pools in the cell.
In recent years small G proteins have become an intensively studied group of regulatory GTP hydrolases involved in cell signaling. More than 100 small G proteins have been identified in eucaryotes from protozoan to human. The small G protein superfamily includes Ras, Rho Rab, Rac, Sarl/Arf and Ran homologs, which take part in numerous and diverse cellular processes, such as gene expression, cytoskeleton reorganization, microtubule organization, and vesicular and nuclear transport. These proteins share a common structural core, described as the G domain, and significant sequence similarity. In this paper we review the available data on G domain structure, together with a detailed analysis of the mechanism of action. We also present small G protein regulators: GTPase activating proteins that bind to a catalytic G domain and increase its low intrinsic hydrolase activity, GTPase dissociation inhibitors that stabilize the GDP-bound, inactive state of G proteins, and guanine nucleotide exchange factors that accelerate nucleotide exchange in response to cellular signals. Additionally, in this paper we describe some aspects of small G protein interactions with down-stream effectors.
The second step in the enzyme-catalyzed hydrolysis of phosphate esters by ribonuclease A (RNase A) was studied using an ab initio quantum-based model of the active site including constrained parts of three critical residues, His-12, His-119, and Lys-41, and a small substrate. The competition between release of the cyclic phosphate intermediate and subsequent hydrolysis following transphosphorylation was explored to determine the electronic factors that contribute to preferential intermediate product release observed experimentally. The structural and energetic results obtained at both the RHF and MP2 levels reveal several contributing factors consistent with experimental observation. Although the intrinsic electronic effects tend to favor hydrolysis slightly with an overall activation free energy of approximately 70 kJ mol(-1), entropic and environmental effects favor release of the cyclic phosphate intermediate over hydrolysis. Exploration of the second, hydrolysis step also revealed interesting similarity with the transphosphorylation step, including the observation of autocatalysis by the substrate. Moreover, both steps of the overall RNase A reaction reveal multiple pathways involving proton transfers to sites of similar proton affinities. The anionic phosphate in both steps can act as a stable proton binding site as protons are moved around the active site throughout the progress of the reaction. These results suggest autocatalysis may be representative of more general behavior in enzymes containing highly charged substrates, especially phosphates.
The elucidation of the structure of the RasGAP complex provides what is perhaps the most detailed link between protein structure and cancer causing mutations. In particular, it is known that mutations of Gln 61 destroy the GTPase activity of the complex, locks the cell in its ON state and thus, can cause cancer. It is entirely unclear however, why this specific mutation is so important. The present work uncovers the elusive role of Gln 61 by computer simulation of the GTPase reaction in Ras, RasGAP and of their mutants. Simulations of the effects of mutations of Gln 61 reproduce the corresponding observed changes in activation energies and allow us to analyze the energy contributions to these effects. It is found that Gln 61 does not operate in a direct chemical way nor by a direct electrostatic or steric interaction with the transition state (TS). Instead, oncogenic mutations of Gln 61 lead to the destruction of the exquisitely preorganized catalytic configuration of the active site of the RasGAP complex. This "allosteric" effect causes a major reduction in the electrostatic stabilization of the TS. Our findings have general relevance to other proteins that control signal transduction processes.
Here, we present MultiProt, a fully automated highly efficient technique to detect multiple structural alignments of protein structures. MultiProt finds the common geometrical cores between input molecules. To date, most methods for multiple alignment start from the pairwise alignment solutions. This may lead to a small overall alignment. In contrast, our method derives multiple alignments from simultaneous superpositions of input molecules. Further, our method does not require that all input molecules participate in the alignment. Actually, it efficiently detects high scoring partial multiple alignments for all possible number of molecules in the input. To demonstrate the power of MultiProt, we provide a number of case studies. First, we demonstrate known multiple alignments of protein structures to illustrate the performance of MultiProt. Next, we present various biological applications. These include: (1) a partial alignment of hinge-bent domains; (2) identification of functional groups of G-proteins; (3) analysis of binding sites; and (4) protein-protein interface alignment. Some applications preserve the sequence order of the residues in the alignment, whereas others are order-independent. It is their residue sequence order-independence that allows application of MultiProt to derive multiple alignments of binding sites and of protein-protein interfaces, making MultiProt an extremely useful structural tool.
Fru-2,6-P2 (fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure-function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2. The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation.
We present results of the modeling for the hydrolysis reaction of guanosine triphosphate (GTP) in the RAS-GAP protein complex using essentially ab initio quantum chemistry methods. One of the approaches considers a supermolecular cluster composed of 150 atoms at a consistent quantum level. Another is a hybrid QM/MM method based on the effective fragment potential technique, which describes interactions between quantum and molecular mechanical subsystems at the ab initio level of the theory. Our results show that the GTP hydrolysis in the RAS-GAP protein complex can be modeled by a substrate-assisted catalytic mechanism. We can locate a configuration on the top of the barrier corresponding to the transition state of the hydrolysis reaction such that the straightforward descents from this point lead either to reactants GTP+H(2)O or to products guanosine diphosphate (GDP)+H(2)PO(4)(-). However, in all calculations such a single-step process is characterized by an activation barrier that is too high. Another possibility is a two-step reaction consistent with formation of an intermediate. Here the Pgamma-O(Pbeta) bond is already broken, but the lytic water molecule is still in the pre-reactive state. We present arguments favoring the assumption that the first step of the GTP hydrolysis reaction in the RAS-GAP protein complex may be assigned to the breaking of the Pgamma-O(Pbeta) bond prior to the creation of the inorganic phosphate.
Controlling the hydrolysis rate of GTP bound to the p21ras protein is crucial for the delicate timing of many biological processes. A few mechanisms were suggested for the hydrolysis of GTP. To gain more insight into the individual elementary events of GTP hydrolysis, we carried out molecular dynamic analysis of wild-type p21ras and some of its mutants. It was recently shown that Ras-related proteins and mutants generally follow a linear free energy relationship (LFER) relating the rate of reaction to the pK(a) of the gamma-phosphate group of the bound GTP, indicating that proton transfer from the attacking water to the GTP is the first elementary event in the GTPase mechanism. However, some exceptions were observed. Thus, the Gly12 --> Aspartic p21ras (G12D) mutant had a very low GTPase activity although its pK(a) was very close to that of the wild-type ras. Here we compared the molecular dynamics (MD) of wild-type Ras and G12D, showing that in the mutant the catalytic water molecule is displaced to a position where proton transfer to GTP is unfavorable. These results suggest that the mechanism of GTPase is indeed composed of an initial proton abstraction from water by the GTP, followed by a nucleophilic attack of the hydroxide ion on the gamma-phosphorus of GTP.
The mechanism of the hydrolysis reaction of guanosine triphosphate (GTP) by the protein complex Ras-GAP (p21(ras) - p120(GAP)) has been modeled by the quantum mechanical-molecular mechanical (QM/MM) and ab initio quantum calculations. Initial geometry configurations have been prompted by atomic coordinates of a structural analog (PDBID:1WQ1). It is shown that the minimum energy reaction path is consistent with an assumption of two-step chemical transformations. At the first stage, a unified motion of Arg789 of GAP, Gln61, Thr35 of Ras, and the lytic water molecule results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low-barrier transition state TS1. At the second stage, Gln61 abstracts and releases protons within the subsystem including Gln61, the lytic water molecule and the gamma-phosphate group of GTP through the corresponding transition state TS2. Direct quantum calculations show that, in this particular environment, the reaction GTP + H(2)O --> GDP + H(2)PO(4) (-) can proceed with reasonable activation barriers of less than 15 kcal/mol at every stage. This conclusion leads to a better understanding of the anticatalytic effect of cancer-causing mutations of Ras, which has been debated in recent years.
The QM/MM MD and free energy simulations show that the dynamics involving a His residue at the P1 site of the substrate may play an important role in substrate-assisted catalysis and specificity for a serine-carboxyl peptidase.
Comparisons of different protein structures are commonly carried out by superimposing the coordinates of the protein backbones or selected parts of the proteins. When the objective is analysis of similarities and differences in the enzyme's active site, there is an inherent problem in using the same domains for the superimposition. In this work we use a comparative approach termed here "Substrate Directed SuperImposition" (SDSI). It entails the superimposition of multiple protein-substrate structures using exclusively the coordinates of the comparable substrates. SDSI has the advantage of unbiased comparison of the active-site environment from the substrate's point of view. Our analysis extends previous usage of similar approaches to comparison of enzyme catalytic machineries. We applied SDSI to various G-protein structures for dissecting the mechanism of the GTPase reaction that controls the signaling activity of this important family. SDSI indicates that dissimilar G-proteins stabilize the transition state of the GTPase reaction similarly and supports the commonality of the critical step in this reaction, the reorientation of the critical arginine and glutamine. Additionally, we ascribe the catalytic inefficiency of the small G-protein Ras to the great flexibility of its active site and downplay the possible catalytic roles of the Lys16 residue in Ras GTPase. SDSI demonstrated that in contrast to all other Gly12 Ras mutants, which are oncogenic, the Gly12-->Pro mutant does not interfere with the catalytic orientation of the critical glutamine. This suggests why this mutant has a higher rate of GTP hydrolysis and is non-transforming. Remarkably, SDSI also revealed similarities in the divergent catalytic machineries of G-proteins and UMP/CMP kinase. Taken together, our results promote the use of SDSI to compare the catalytic machineries of both similar and different classes of enzymes.
We have introduced two types of mutations into cDNAs that encode the α subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase. The arginine residue (Arg¹⁸⁷) that is the presumed site of ADP-ribosylation of Gsα by cholera toxin has been changed to Ala, Glu, or Lys. The rate constant for hydrolysis of GTP by all of these mutants is reduced approximately 100-fold compared with the wild-type protein. As predicted from this change, these proteins activate adenylyl cyclase constitutively in the presence of GTP. Despite these substitutions, cholera toxin still catalyzes the incorporation of 0.2-0.3 mol of ADP-ribose/mol of mutant α subunit.
The sequence near the carboxyl terminus of Gsα was altered to resemble those in Giα polypeptides, which are substrates for pertussis toxin. Despite this change, the mutant protein is a poor substrate for pertussis toxin. Although this protein has unaltered rates of GDP dissociation and GTP hydrolysis, its ability to activate adenylyl cyclase in the presence of GTP is enhanced by 3-fold when compared with the wild-type protein but only when these assays are performed after reconstitution of Gsα into cyc⁻ (Gsα-deficient) S49 cell membranes.
Ras proteins are guanine-nucleotide binding proteins that have a low intrinsic GTPase activity that is enhanced 105-fold by the GTPase-activating proteins (GAPs) p120-GAP and neurofibromin. Comparison of the primary sequences of RasGAPs
shows two invariant arginine residues (Arg1276 and Arg1391 of neurofibromin). In this study, site-directed mutagenesis was used to change each of these residues in the catalytic domain
of neurofibromin (NF1-334) to alanine. The ability of the mutant proteins to bind to Ras·GTP and to stimulate their intrinsic
GTPase rate was then determined by kinetic methods under single turnover conditions using a fluorescent analogue of GTP. The
separate contributions of each of these residues to catalysis and binding affinity to Ras were measured. Both the R1276A and
the R1391A mutant NF1-334 proteins were 1000-fold less active than wild-type NF1-334 in activating the GTPase when measured
at saturating concentrations. In contrast, there was only a minor effect of either mutation on NF1-334 affinity for wild-type
Ha-Ras. These data are consistent with both arginines being required for efficient catalysis. Neither arginine is absolutely
essential, because the mutant NF1-334 proteins increase the intrinsic Ras·GTPase by at least 100-fold. The roles of Arg1276 and Arg1391 in neurofibromin are consistent with proposals based on the recently published x-ray structure of p120-GAP complexed with
Ras.
Crystals of Ha-Ras p21 with caged GTP at the active site have been used to investigate the conformational changes of p21 on GTP hydrolysis. The structure of the short-lived p21.GTP complex was determined by Laue diffraction methods. After GTP hydrolysis, substantial structural changes occur in the parts of the molecule implicated in the interaction with GTPase-activating protein. The trigger for this process seems to be a change in coordination of the active-site Mg2+ ion as a result of loss of the gamma-phosphate of GTP.
We have introduced two types of mutations into cDNAs that encode the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase. The arginine residue (Arg187) that is the presumed site of ADP-ribosylation of Gs alpha by cholera toxin has been changed to Ala, Glu, or Lys. The rate constant for hydrolysis of GTP by all of these mutants is reduced approximately 100-fold compared with the wild-type protein. As predicted from this change, these proteins activate adenylyl cyclase constitutively in the presence of GTP. Despite these substitutions, cholera toxin still catalyzes the incorporation of 0.2-0.3 mol of ADP-ribose/mol of mutant alpha subunit. The sequence near the carboxyl terminus of Gs alpha was altered to resemble those in Gi alpha polypeptides, which are substrates for pertussis toxin. Despite this change, the mutant protein is a poor substrate for pertussis toxin. Although this protein has unaltered rates of GDP dissociation and GTP hydrolysis, its ability to activate adenylyl cyclase in the presence of GTP is enhanced by 3-fold when compared with the wild-type protein but only when these assays are performed after reconstitution of Gs alpha into cyc- (Gs alpha-deficient) S49 cell membranes.
Despite many advances in understanding the structure and function of GTP-binding proteins the mechanism by which these molecules switch from the GTP-bound on-state to the GDP-bound off-state is still poorly understood. Theoretical studies suggest that the activation of the nucleophilic water which hydrolyzes GTP needs a general base. Such a base could not be located in any of the many GTP-binding proteins. Here we present a unique type of linear free energy relationships that not only supports a mechanism for p21ras in which the substrate GTP itself acts as the catalytic base driving the GTPase reaction but can also help to explain why certain mutants of p21ras are oncogenic and others are not.
The three-dimensional structure of the complex between human H-Ras bound to guanosine diphosphate and the guanosine triphosphatase
(GTPase)–activating domain of the human GTPase-activating protein p120GAP (GAP-334) in the presence of aluminum fluoride was solved at a resolution of 2.5 angstroms. The structure shows the partly
hydrophilic and partly hydrophobic nature of the communication between the two molecules, which explains the sensitivity of
the interaction toward both salts and lipids. An arginine side chain (arginine-789) of GAP-334 is supplied into the active
site of Ras to neutralize developing charges in the transition state. The switch II region of Ras is stabilized by GAP-334,
thus allowing glutamine-61 of Ras, mutation of which activates the oncogenic potential, to participate in catalysis. The structural
arrangement in the active site is consistent with a mostly associative mechanism of phosphoryl transfer and provides an explanation
for the activation of Ras by glycine-12 and glutamine-61 mutations. Glycine-12 in the transition state mimic is within van
der Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras, and even its mutation to alanine would disturb
the arrangements of residues in the transition state.
RasGAPs supply a catalytic residue, termed the arginine finger,into the active site of Ras thereby stabilizing the transition state of the GTPase reaction and increasing the reaction rate by more than one thousand-fold, in good agreement with the structure of the Ras.RasGAP complex.
The origin of the catalytic power of enzymes is discussed, paying attention to evolutionary constraints. It is pointed out that enzyme catalysis reflects energy contributions that cannot be determined uniquely by current experimental approaches without augmenting the analysis by computer simulation studies. The use of energy considerations and computer simulations allows one to exclude many of the popular proposals for the way enzymes work. It appears that the standard approaches used by organic chemists to catalyze reactions in solutions are not used by enzymes. This point is illustrated by considering the desolvation hypothesis and showing that it cannot account for a large increase in kcat relative to the corresponding kcage for the reference reaction in a solvent cage. The problems associated with other frequently invoked mechanisms also are outlined. Furthermore, it is pointed out that mutation studies are inconsistent with ground state destabilization mechanisms. After considering factors that were not optimized by evolution, we review computer simulation studies that reproduced the overall catalytic effect of different enzymes. These studies pointed toward electrostatic effects as the most important catalytic contributions. The nature of this electrostatic stabilization mechanism is far from being obvious because the electrostatic interaction between the reacting system and the surrounding area is similar in enzymes and in solution. However, the difference is that enzymes have a preorganized dipolar environment that does not have to pay the reorganization energy for stabilizing the relevant transition states. Apparently, the catalytic power of enzymes is stored in their folding energy in the form of the preorganized polar environment.
The Rho-related small GTP-binding protein Cdc42 has a low intrinsic GTPase activity that is significantly enhanced by its specific GTPase-activating protein, Cdc42GAP. In this report, we present the tertiary structure for the aluminum fluoride-promoted complex between Cdc42 and a catalytically active domain of Cdc42GAP as well as the complex between Cdc42 and the catalytically compromised Cdc42GAP(R305A) mutant. These structures, which mimic the transition state for the GTP hydrolytic reaction, show the presence of an AIF3 molecule, as was seen for the corresponding Ras-p120RasGAP complex, but in contrast to what has been reported for the Rho-Cdc42GAP complex or for heterotrimeric G protein alpha subunits, where AIF4- was observed. The Cdc42GAP stabilizes both the switch I and switch II domains of Cdc42 and contributes a highly conserved arginine (Arg 305) to the active site. Comparison of the structures for the wild type and mutant Cdc42GAP complexes provides important insights into the GAP-catalyzed GTP hydrolytic reaction.
The formation of a complex between p21(ras) and GAP accelerates the GTPase reaction of p21(ras) and terminates the signal for cell proliferation. The understanding of this rate acceleration is important for the elucidation of the role of Ras mutants in tumor formation. In principle there are two main options for the origin of the effect of GAP. One is a direct electrostatic interaction between the residues of GAP and the transition state of the Ras-GAP complex and the other is a GAP-induced shift of the structure of Ras to a configuration that increases the stabilization of the transition state. This work examines the relative importance of these options by computer simulations of the catalytic effect of Ras. The simulations use the empirical valence bond (EVB) method to study the GTPase reaction along the alternative associative and dissociative paths. This approach reproduces the trend in the overall experimentally observed catalytic effect of GAP: the calculated effect is 7 +/- 3 kcal/mol as compared to the observed effect of approximately 6.6 kcal/mol. Furthermore, the calculated effect of mutating Arg789 to a nonpolar residue is 3-4 kcal/mol as compared to the observed effect of 4.5 kcal/mol for the Arg789Ala mutation. It is concluded, in agreement with previous proposals, that the effect of Arg789 is associated with its direct interaction with the transition state charge distribution. However, calculations that use the coordinates of Ras from the Ras-GAP complex (referred to here as Ras') reproduce a significant catalytic effect relative to the Ras coordinates. This indicates that part of the effect of GAP involves a stabilization of a catalytic configuration of Ras. This configuration increases the positive electrostatic potential on the beta-phosphate (relative to the corresponding situation in the free Ras). In other words, GAP stabilizes the GDP bound configuration of Ras relative to that of the GTP-bound conformation. The elusive oncogenic effect of mutating Gln61 is also explored. The calculated effect of such mutations in the Ras-GAP complex are found to be small, while the observed effect is very large (8.7 kcal/mol). Since the Ras is locked in its Ras-GAP configuration in our simulations, we conclude that the oncogenic effect of mutation of Gln61 is indirect and is associated most probably with the structural changes of Ras upon forming the Ras-GAP complex. In view of these and the results for the Ras' we conclude that GAP activates Ras by both direct electrostatic stabilization of the transition state and an indirect allosteric effect that stabilizes the GDP-bound form. The present study also explored the feasibility of the associative and dissociative mechanism in the GTPase reaction of Ras. It is concluded that the reaction is most likely to involve an associative mechanism.
RasGAPs supply a catalytic residue, termed the arginine finger,into the active site of Ras thereby stabilizing the transition state of the GTPase reaction and increasing the reaction rate by more than one thousand-fold, in good agreement with the structure of the Ras.RasGAP complex.
The active GTP-bound form of p21(ras) is converted to the biologically inactive GDP-bound form by enzymatic hydrolysis and this function serves to regulate the wild-type ras protein. The side chain of the amino acid at position 61 may play a key role in this hydrolysis of GTP by p21. Experimental studies that define properties of the Q61E mutant of p21(H-ras) are presented along with supporting molecular dynamics simulations. We find that under saturating concentrations of GTP the Q61E mutant of p21(H-ras) has a 20-fold greater rate of intrinsic hydrolysis (k(cat) = 0.57 min(-1)) than the wild type. The affinity of the Q61E variant for GTP (K-d = 115 mu M) is much lower than that of the wild type. GTPase activating protein does not activate the variant. From molecular dynamics simulations, we find that both the wild type and Q61E mutant have the residue 61 side chain in transient contact with a water molecule that is well-positioned for hydrolytic attack on the gamma phosphate. Thr-35 also is found to form a transient hydrogen bond with this critical water. These elements may define the catalytic complex for hydrolysis of the GTP [Pai et. al. (1990) EMBO J. 9, 2351]. Similarly, the G12P mutant, which also has an intrinsic hydrolysis rate similar to the wild type, is found to form the same complex in simulation. In contrast, molecular dynamics analysis of the mutants G12R, G12V, and Q61L, which have much lower intrinsic rates than the wild-type p21, do not show this complex. Thus, the experimental data for intrinsic hydrolysis rates and the molecular dynamics simulations support the view that the residue 61 side chain is involved in activating a water molecule in the GTP hydrolysis mechanism.
Despite many advances in understanding the structure and function of GTP-binding proteins the mechanism by which these molecules switch from the GTP-bound on-state to the GDP-bound off-state is still poorly understood. Theoretical studies suggest that the activation of the nucleophilic water which hydrolyzes GTP needs a general base. Such a base could not be located in any of the many GTP-binding proteins. Here we present a unique type of linear free energy relationships that not only supports a mechanism for p21rasin which the substrate GTP itself acts as the catalytic base driving the GTPase reaction but can also help to explain why certain mutants of p21ras are oncogenic and others are not.
The X-ray structures of the guanine nucleotide binding domains (amino acids 1–166) of five mutants of the H-ras oncogene product p21 were determined. The mutations described are Gly-12→Arg, Gly-12→Val, Gln-61→His, Gln-61→Leu, which are all oncogenic, and the effector region mutant Asp-38→Glu. The resolutions of the crystal structures range from 2.0 to 2.6 Å. Cellular and mutant p21 proteins are almost identical, and the only significant differences are seen in loop L4 and in the vicinity of the γ-phosphate. For the Gly-12 mutants the larger side chains interfere with GTP binding and/or hydrolysis. Gln-61 in cellular p21 adopts a conformation where it is able to catalyze GTP hydrolysis. This conformation has not been found for the mutants of Gln-61. Furthermore, Leu-61 cannot activate the nucleophilic water because of the chemical nature of its side chain. The D38E mutation preserves its ability to bind GAP.
Determination of specific GTPase (EC 3.6.1.--) activity in turkey erythrocyte membranes was achieved using low concentration of GTP (0.25 muM), inhibition of nonspecific nucleoside triphosphatases by adenosine 5'(beta,gamma-imino-triphosphate (App(NH)p) and suppression of the transfer of gamma-32P from GTP to ADP with an ATP regeneration system. Under these conditions catacholamines caused a 30--70% increase in GTP hydrolysis. The stimulation of GTPase activity by catecholamines required the presence of Mg2+ or Mn2+. DIfferent batches of membranes revealed the following specific activities (pmol 32Pi/mg protein min): basal GTPase (determined in the absence of catecholamine), 6-- 11; catecholamine-stimulated TTPase, 3--7; and residual non-specific NTPase 3--5. The stimulation of GTPase activity by catecholamines fulfilled the stereospecific requirements of the beta-adrenergic receptor, and was inhibited by propranolol. The concentrations of DL-isoproterenol which half-maximally activated the GTPase and adenylate cyclase were 1 and 1.2 muM, respectively. The following findings indicate that the catecholamine-stimulated GTPase is independent of the catalytic production of cyclic AMP by the adenylate cyclase. Addition of cyclic AMP to the GTPase assay did not change the rate of GTP hydrolysis. Furthermore, treatment of the membrane with N-ethylmaleimide (MalNEt) at 0 degrees C which caused 98% inhibition of the adenylate cyclase, had no effect on the catecholamine-stimulated GTPase. The affinity and specificity for GTP in the GTPase reactions are similar to those previously reported for the stimulation of the adenylate cyclase. The apparent Km for GTP in the basal and the catecholamine-stimulated GTPase reaction was 0.1 muM. These GTPase activities were inhibited by ITP but not by CTP and UTP. It is proposed that a catecholamine-stimulated GTPase is a component of the turkey erythrocyte adenylate cyclase system.
Treatment of turkey erthrocyte membranes with cholera toxin caused an enhancement of the basal and catecholamine-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activities. Both of these activities required the presence of GTP. The toxin effect on the adenylate cyclase activity concided with an inhibition of the catecholamine-stimulated guanosinetriphosphatase activity. Inhibition of the guanosinetriphosphatase, as well as enhancement of the adenylate cyclase activity, showed the same dependence on cholera toxin concentrations, and the effect of the toxin on both activities was dependent on the presence of NAD.
It is proposed that continuous GTP hydrolysis at the regulatory guanyl nucleotide site is an essential turn-off mechanism, terminating activation of the adenylate cyclase. Cholera toxin inhibits the turn-off guanosinetriphosphatase reaction and thereby causes activation of the adenylate cyclase. According to this mechanism GTP should activate the toxin-treated preparation of adenylate cyclase, as does the hydrolysis-resistant analog guanosine 5′-(β,γ-immino)triphosphate [Gpp(NH)p]. Indeed, the toxin-treated adenylate cyclase was maximally activated, in the presence of isoproternol, by either GTP or Gpp(NH)p, while adenylate cyclase not treated with toxin was stimulated by hormone plus GTP to only one-fifth of the activity achieved with hormone plus Gpp(NH)p. Furthermore, the toxin-treated adenylate cyclase activated by isoproterenol plus GTP remained active for and extended period (half-time of 3 min) upon subsequent addition of the β-adrenergic blocker, propranolol. The native enzyme, however, was refractory to propranolol only if activated by Gpp(NH)p but not by GTP.
It has recently been suggested that adenylate cyclase activity is controlled by a regulatory cycle consisting of two reactions: a hormone induced formation of the active adenylate cyclase-GTP complex, and a subsequent turn-off reaction in which hydrolysis of the bound nucleotide reverts the system to the inactive state. To test this model each of the two reactions was measured separately and their rate constants were used to estimate the steady state adenylate cyclase and GTPase activities. The first order rate constants were kon = 3 min-1 for the activation reaction and koff = 15 min-1 for the turn-off reaction. Substitution of these rate constants in the steady state equation of the regulatory cycle gave values of hormone stimulated adenylate cyclase and GTPase activities similar to those determined by direct measurements. Treatment of the adenylate cyclase with cholera toxin caused a decrease of 96% in the rate constant of the turn-off reaction. In this case too the activities calculated from the steady state equation were in good agreement with those determined directly.
The fate of the guanyl nucleotide bound to the regulatory site of adenylate cyclase was studied on a preparation of turkey erythrocyte membranes that was incubated with [3H]GTP plus isoproterenol and subsequently washed to remove hormone and free guanyl nucleotide. Further incubation of this preparation in the presence of beta-adrenergic agonists resulted in the release from the membrane of tritiated nucleotide, identified as [3H]GDP. The catecholamine-induced release of [3H]GDP was increased 2 to 3 times in the presence of the unlabeled guanyl nucleotides GTP, guanosine 5'-(beta,gamma-imino)triphosphate [gpp(NH)p], GDP, and GMP, whereas adenine nucleotides had little effect. In the presence of Gpp(NH)p, isoproterenol induced the release of [3H]GDP and the activation of adenylate cyclase, both effects following similar time courses. The findings indicate that the inactive adenylate cyclase possesses tightly bound (GDP, produced by the hydrolysis of GTP at the regulatory site. The hormone stimulates adenylate cyclase activity by inducing an "opening" of the guanyl nucleotide site, resulting in dissociation of the bound GDP and binding of the activating guanosine triphosphate.
Mutations in codon 12, 13, or 61 of one of the three ras genes, H-ras, K-ras, and N-ras, convert these genes into active oncogenes. Rapid assays for the detection of these point mutations have been developed recently and used to investigate the role mutated ras genes play in the pathogenesis of human tumors. It appeared that ras gene mutations can be found in a variety of tumor types, although the incidence varies greatly. The highest incidences are found in adenocarcinomas of the pancreas (90%), the colon (50%), and the lung (30%); in thyroid tumors (50%); and in myeloid leukemia (30%). For some tumor types a relationship may exist between the presence of a ras mutation and clinical or histopathological features of the tumor. There is some evidence that environmental agents may be involved in the induction of the mutations.
A subset of growth hormone-secreting human pituitary tumours carries somatic mutations that inhibit GTPase activity of a G protein alpha chain, alpha(s). The resulting activation of adenylyl cyclase bypasses the cells' normal requirement for trophic hormone. Amino acids substituted in the putative gsp oncogene identify a domain of G protein alpha-chains required for intrinsic ability to hydrolyse GTP. This domain may serve as a built-in counter-part of the separate GTPase-activating proteins required for GTP hydrolysis by small GTP-binding proteins such as p21ras.
The rate of GTP hydrolysis on p21ras is accelerated by approximately 10(5) times by the catalytic domains of GTPase-activating proteins (GAPs), p120-GAP (GAP-344) or neurofibromin (NF1-334). The kinetic mechanism of this activation has been investigated by following the release of inorganic phosphate (Pi), using a fluorescent probe that is sensitive to Pi [Brune, M., Hunter, J., Corrie, J. E. T., & Webb, M. R. (1994) Biochemistry 33, 8262-8271]. Measurements were made in real time with a stopped-flow apparatus, in which the p21ras complex with the 2',3'-methanthraniloyl analogue of GTP (mantGTP) was mixed with the GAP in the presence of this Pi probe. The results show that Pi release is fast and that the overall hydrolysis is controlled by the cleavage itself or a conformational change preceding the cleavage. The time courses were single exponentials over a range of [GAP-344] and were modeled to show that a single step controlled Pi release. The maximum rate constant was 15 s-1 (all data at 30 degrees C, pH 7.6, low ionic strength) in experiments in which GAP-344 underwent a single turnover, compared with 5 s-1 for multiple-turnover experiments, and possible causes of this discrepancy were investigated and discussed. With NF1-334 the time courses were more complex, showing a lag prior to rapid release of Pi. The results were consistent with a Kd of 0.04 microM for NF1-344 affinity is some 3 orders of magnitude tighter than that of GAP-344.(ABSTRACT TRUNCATED AT 250 WORDS)
Ras p21 plays a major role in the control of cell growth, and oncogenic mutations of this protein have been found in human cancers. Unfortunately, the detailed mode of action of Ras p21 is still unclear, in spite of the great interest in this protein and the availability of its X-ray crystal structure. In particular, mutagenesis studies of different active site residues could not identify the general base for GTP hydrolysis. Here we tackle this question using a computer simulation approach with clear and reliable energy considerations and conclude that the most likely general base is the bound GTP itself. Obviously, the identification of such a general base cannot be easily accomplished by mutagenesis experiments.
Orotic acid is decarboxylated with a half-time (t1/2) of 78 million years in neutral aqueous solution at room temperature,
as indicated by reactions in quartz tubes at elevated temperatures. Spontaneous hydrolysis of phosphodiester bonds, such as
those present in the backbone of DNA, proceeds even more slowly at high temperatures, but the heat of activation is less positive,
so that dimethyl phosphate is hydrolyzed with a t1/2 of 130,000 years in neutral solution at room temperature. These values
extend the known range of spontaneous rate constants for reactions that are also susceptible to catalysis by enzymes to more
than 14 orders of magnitude. Values of the second-order rate constant kcat/Km for the corresponding enzyme reactions are confined
to a range of only 600-fold, in contrast. Orotidine 5'-phosphate decarboxylase, an extremely proficient enzyme, enhances the
rate of reaction by a factor of 10(17) and is estimated to bind the altered substrate in the transition state with a dissociation
constant of less than 5 x 10(-24) M.
The mechanism of the hydrolysis of GTP by p21N-ras and its activation by the catalytic domain of p120 GTPase activating protein (GAP) have been studied using a combination of chemical and fluorescence measurements with the fluorescent GTP analogue, 2'(3')-O-(N-methylanthraniloyl)GTP (mantGTP). Since the concentration of active p21 is important in these measurements, various assays for both total protein and active p21 were investigated. All assays gave good agreement except the filter binding assay of [3H]-GDP bound to p21, which gave values of 35-40% compared to the other methods. Concentrations of p21 were thus based on the absorbance of the mant-chromophore of the p21-mant-nucleotide complexes. The rate constants of the elementary steps of the p21 intrinsic GTPase activity and the GAP activated activity were similar between GTP and mantGTP. Incubation of a stoichiometric complex of p21.mantGTP results in a biphasic decrease in fluorescence. The second phase occurs with the same rate constant as the cleavage step and is accelerated by GAP. No other steps of the mechanism are affected by GAP. Incubation of a stoichiometric complex of p21.mantGpp[NH]p also results in a biphasic decrease in fluorescence even though cleavage does not occur. This is interpreted that the cleavage step of p21.GTP is preceded by and controlled by an isomerization of the p21.GTP complex. GAP accelerates the rate constant of the second fluorescence phase occurring with p21.mantGpp[NH]p. This result shows that GAP accelerates the proposed isomerization which limits GTP cleavage rather than the cleavage step itself.
Previous studies of the GTPase reaction catalyzed by p21ras have indicated that the logarithm of the observed reaction rate and the pKa of the bound GTP are correlated by the Brønsted relationship log(kcat) = beta pKa + A. While most of the Ras mutants display a Brønsted slope beta of 2.1, a small set of oncogenic mutants exhibit a beta of > > 1. On the other hand, it was found that the corresponding Brønsted slope for the GTPase reaction of p21ras in the presence of GTPase Activating Protein (GAP) is about beta = 4.9. The present work explores the basis for such linear free energy relationships (LFERs) in general and applies these concepts to p21ras and related systems. It is demonstrated that the optimal way to analyze LFER is by using Marcus type parabolas that represent the reactant, intermediate, and product state of the reaction in a relevant energy diagram. The observed LFER is used to analyze the actual free energy surface and reaction path of the intrinsic GTPase reaction in p21ras. From this, a model reaction profile can be constructed that explains how a LFER can arise and also how the different observed Brønsted coefficients can be rationalized. This analysis is augmented by solvent isotope effect studies. It is pointed out that the overall activation barrier reflects the energy of the proton transfer (PT) step, although this step does not include the actual transition state of the hydrolysis reaction. The proposed GTP as a base mechanism is compared to a recently proposed reaction scheme where Gln61 serves as a proton shuttle in a concerted mechanism. It is shown by unique energy considerations that the concerted mechanism is unlikely. Other alternative mechanisms are also considered, and their consistency with the observed LFER and other factors is discussed. Finally, we analyze the observed LFER for the GTPase reaction of p21ras in the presence of GAP and discuss its relevance for the mechanism of GAP activation.
Controlling the hydrolysis rate of GTP bound to guanine nucleotide binding proteins is crucial for the right timing of many biological processes. Theoretical, structural, and functional studies have demonstrated that in p21ras the substrate of the reaction, GTP itself, plays a central role by acting as the base catalyst. This substrate-assisted reaction mechanism was analyzed with the help of linear free energy relationships (LFERs). Here we present experimental data that further support the proposed mechanism. We extend the LFER analysis to a wide range of oncogenic as well as nontransforming Ras mutants. It is illustrated that almost all Ras variants follow the observed LFER and thus also the same reaction path. Further, the reduced GTPase reaction rate that characterizes the oncogenic effect of many of the p21 mutants found in human tumors seems to be a consequence of a slightly reduced pKa of the gamma-phosphate group of bound GTP. Factors causing a pKa deviation of just 0.5 unit are enough to slow the intrinsic GTPase reaction rate significantly, and the system may exhibit as a consequence of this an oncogenic potential. Interestingly, we also found oncogenic mutations that do not follow the regular LFER. This suggests that the oncogenic effect of distinct Ras mutants has a different physical origin. The results presented might aid in the design of drugs aimed at reactivating the GTPase reaction of many oncogenic p21ras mutants. We also analyzed the stimulated GTPase reaction of p21ras by the GTPase activating protein (GAP) and the GTPase reaction of Rap1A, a Ras-related GTP binding protein, with similar approaches. The corresponding results indicate that the GAP-stimulated GTPase as well as the Rap1A-catalyzed reaction seem to follow the same substrate-assisted reaction mechanism. However, the correlation coefficient for the GAP-catalyzed reaction is different from the corresponding coefficient for the intrinsic reaction. While the intrinsic reaction exhibits a Brønsted slope of beta = 2.1, the corresponding value for the GAP-activated reaction is beta = 4.9.
RGS proteins are GTPase activators for heterotrimeric G proteins. We report here the 2.8 A resolution crystal structure of the RGS protein RGS4 complexed with G(i alpha1)-Mg2+-GDP-AlF4 . Only the core domain of RGS4 is visible in the crystal. The core domain binds to the three switch regions of G(i alpha1), but does not contribute catalytic residues that directly interact with either GDP or AlF4-. Therefore, RGS4 appears to catalyze rapid hydrolysis of GTP primarily by stabilizing the switch regions of G(i alpha1), although the conserved Asn-128 from RGS4 could also play a catalytic role by interacting with the hydrolytic water molecule or the side chain of Gln-204. The binding site for RGS4 on G(i alpha1) is also consistent with the activity of RGS proteins as antagonists of G(alpha) effectors.
The backbone 1H, 13C, and 15N resonances of the c-Ha-Ras protein [a truncated version consisting of residues 1-171, Ras(1-171)] bound with GMPPNP (a slowly hydrolyzable analogue of GTP) were assigned and compared with those of the GDP-bound Ras(1-171). The backbone amide resonances of amino acid residues 10-13, 21, 31-39, 57-64, and 71 of Ras(1-171).GMPPNP, but not those of Ras(1-171).GDP, were extremely broadened, whereas other residues of Ras(1-171).GMPPNP exhibited amide resonances nearly as sharp as those of Ras(1-171). GDP. The residues exhibiting the extreme broadening, except for residues 21 and 71, are localized in three functional loop regions [loops L1, L2 (switch I), and L4 (switch II)], which are involved in hydrolysis of GTP and interactions with other proteins. From the temperature and magnetic field strength dependencies of the backbone amide resonance intensities, the extreme broadening was ascribed to the exchange at an intermediate rate on the NMR time scale. It was shown that the Ras(1-171) protein bound with GTP or GTPgammaS (another slowly hydrolyzable analogue of GTP) exhibits the same type of broadening. Therefore, it is a characteristic feature of the GTP-bound form of Ras that the L1, L2, and L4 loop regions, but not other regions, are in a rather slow interconversion between two or more stable conformers. This phenomenon, termed a "regional polysterism", of these loop regions may be related with their multifunctionality: the GTP-dependent interactions with several downstream target groups such as the Raf and RalGDS families and also with the GTPase activating protein (GAP) family. In fact, the binding of Ras(1-171).GMPPNP with the Ras-binding domain (residues 51-131) of c-Raf-1 was shown to eliminate the regional polysterism nearly completely. It was indicated, therefore, that each target/regulator selects its appropriate conformer among those presented by the "polysteric" binding interface of Ras. As the downstream target groups exhibit no apparent sequence homology to each other, it is possible that one target group prefers a conformer different from that preferred by another group. The involvement of loop L1 in the regional polysterism might suggest that the negative regulators, GAPs, bind to the polysteric binding interface (loops L2 and L4) of Ras and cooperatively select a conformer suitable for transition of the GTPase catalytic center, involving loops L1 and L4, into the highly active state.
This review is concerned with the structures and mechanisms of a superfamily of regulatory GTP hydrolases (G proteins). G proteins include Ras and its close homologs, translation elongation factors, and heterotrimeric G proteins. These proteins share a common structural core, exemplified by that of p21ras (Ras), and significant sequence identity, suggesting a common evolutionary origin. Three-dimensional structures of members of the G protein superfamily are considered in light of other biochemical findings about the function of these proteins. Relationships among G protein structures are discussed, and factors contributing to their low intrinsic rate of GTP hydrolysis are considered. Comparison of GTP- and GDP-bound conformations of G proteins reveals how specific contacts between the gamma-phosphate of GTP and the switch II region stabilize potential effector-binding sites and how GTP hydrolysis results in collapse (or reordering) of these surfaces. A GTPase-activating protein probably binds to and stabilizes the conformation of its cognate G protein that recognizes the transition state for hydrolysis, and may insert a catalytic residue into the G protein active site. Inhibitors of nucleotide release, such as the beta gamma subunit of a heterotrimeric G protein, bind selectively to and stabilize the GDP-bound state. Release factors, such as the translation elongation factor, Ts, also recognize the switch regions and destabilize the Mg(2+)-binding site, thereby promoting GDP release. G protein-coupled receptors are expected to operate by a somewhat different mechanism, given that the GDP-bound form of many G protein alpha subunits does not contain bound Mg2+.
Signaling by guanine-nucleotide-binding proteins (G-proteins) occurs when they are charged with GTP, while hydrolysis of the bound nucleotide turns the signaling off. Despite a wealth of biochemical and structural information, the mechanism of GTP hydrolysis by G-proteins remains controversial. We have employed substrate-assisted catalysis as a novel approach to study catalysis by G-proteins. In these studies, we have used diaminobenzophenone-phosphonoamidate-GTP, a unique GTP analog bearing the functional groups that are missing in the GTPase-deficient [Leu227]G(s alpha) mutant. This mutant, found in various human tumors, fails to hydrolyze GTP for an extended period. In contrast, the GTP analog is hydrolyzed by this mutant and by the wild-type enzyme at the same rate. On the other hand, modification of G(s alpha) by cholera toxin, which catalyses ADP-ribosylation of Arg201 of G(s alpha), decreased the rates of hydrolysis of both GTP and its analog by 95%. These results attest to the specificity of the GTP analog as a unique substrate for the [Leu227]G(s alpha) mutant and to the essential role of Gln227 in GTP hydrolysis. Furthermore, the finding that the GTP analog was hydrolyzed at the same rate as GTP by the wild-type enzyme, favors a model in which formation of a pentavalent transition state intermediate, presumably stabilized by the catalytic glutamine, is not the rate-limiting step of the GTPase reaction.
Recent three-dimensional structures of phosphoryl transfer enzymes in their aluminum fluoride bound state and corresponding biochemical data have shown how diverse biological problems can be investigated using this small inorganic molecule.
A normal mode and energy minimization of ras p21 is used to determine the flexibility of the protein and the origin of the conformational differences between GTP and GDP-bound forms. To preserve the integrity of the structures, a hydration shell of water molecules was included as part of the system. Certain low-frequency modes were found to have high involvement coefficients with the conformational transition between the GTP and GDP-bound structures; the involvement coefficients of some of the modes increase when the gamma-phosphate group is removed. Two unstable modes that appear in the GTP-bound structure upon deletion of the gamma-phosphate group were determined and shown to have dominant contributions in the regions of switch I and switch II; there was also a significant displacement of loop 1. The initial motion in these regions is predicted by the modes to be approximately perpendicular to the direction of the transition from the GTP-bound state to the GDP-bound state. The overall conformational change in the switch I and II regions involves rearrangements of the protein backbone within these regions, rather than rigid body motion. Differences in the low-frequency modes of the GTP and GDP-bound forms appear to play a role in ligand binding. A coupling between the helix alpha3 position and the deletion of the gamma-phosphate group may be involved in the interaction with GAP. The oncogenic mutation G12D leads to a global increase in the rigidity of the protein. Thus, the mutant is likely to have a higher barrier for the conformational change to the inactive form; this would slow the transition and could be related to its oncogenic properties.
Stimulation of the intrinsic GTPase activity of GTP-binding proteins by GTPase-activating proteins (GAPs) is a basic principle of GTP-binding-protein downregulation. Recently, the molecular mechanism behind this reaction has been elucidated by studies on Ras and Rho, and their respective GAPs. The basic features involve stabilizing the existing catalytic machinery and supplementing it by an external arginine residue. This represents a novel mechanism for enzyme active-site formation.
Hydrolysis of GTP, bound to members of the G-protein superfamily, terminates their downstream signaling activity. A conserved glutamine serves a critical role in this pivotal guanosine triphosphatase (GTPase) reaction. However, the role of the catalytic glutamine in GTP hydrolysis is still not well understood. We have employed substrate-assisted catalysis to probe the catalytic mechanism of Gs alpha using GTP analogues. These GTP analogues, each having different functional groups, were designed to support or refute particular putative GTPase mechanisms. We have found that a hydrogen donor group, in close proximity to the gamma-phosphate of GTP, is necessary and sufficient to substitute for the function of the catalytic glutamine in the GTPase reaction.
The Rho family of small GTP-binding proteins are downregulated by an intrinsic GTPase, which is enhanced by GTPase-activating proteins (GAPs). RhoGAPs contain a single conserved arginine residue that has been proposed to be involved in catalysis. Here, the role of this arginine has been elucidated by mutagenesis followed by determination of catalytic and equilibrium binding constants using single-turnover kinetics, isothermal titration calorimetry, and scintillation proximity assays. The turnover numbers for wild-type, R282A, and R282K RhoGAPs were 5.4, 0.023, and 0.010 s-1, respectively. Thus, the function of this arginine could not be replaced by lysine or alanine. Nevertheless, the R282A mutation had a minimal effect on the binding affinity of RhoGAP for either Rho. GTP or Rho.GMPPNP, which confirms the importance of the arginine residue for catalysis as opposed to formation of the protein-protein complex. The R282A mutant RhoGAP still increased the hydrolysis rate of Rho.GTP by 160-fold, whereas the wild-type enzyme increased it by 38000-fold. We conclude that this arginine contributes half of the total reduction of activation energy of catalysis. In the presence of aluminum fluoride, the R282A mutant RhoGAP binds almost as well as the wild type to Rho.GDP, demonstrating that the conserved arginine is not required for this interaction. The affinity of wild-type RhoGAP for the triphosphate form of Rho is similar to that for Rho.GDP with aluminum fluoride. These last two observations show that this complex is not associated with the free energy changes expected for the transition state, although the Rho.GDP.AlF4-.RhoGAP complex might well be a close structural approximation.
Disease-causing mutations often reveal key pathways of physiologic regulation and their underlying molecular mechanisms. Mutations in the trimeric guanine nucleotide-binding proteins (G proteins), which relay signals initiated by photons, odorants, and a host of hormones and neurotransmitters, cause many diseases. For the most part, the diseases are confined to a set of fascinating but rare endocrine disorders (Table 1).1 A recent study suggests that mutations in G proteins can also lead to essential hypertension.2 If this study is correct, hypertension may be one of several common disorders caused by defects in this ubiquitous family of signaling molecules. This review focuses . . .
Interest in the guanosine triphosphatase (GTPase) reaction of Ras as a molecular drug target stems from the observation that, in a large number of human tumors, Ras is characteristically mutated at codons 12 or 61, more rarely 13. Impaired GTPase activity, even in the presence of GTPase activating proteins, has been found to be the biochemical reason behind the oncogenicity of most Gly12/Gln61 mutations, thus preventing Ras from being switched off. Therefore, these oncogenic Ras mutants remain constitutively activated and contribute to the neoplastic phenotype of tumor cells. Here, we show that the guanosine 5'-triphosphate (GTP) analogue diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) is hydrolyzed by wild-type Ras but more efficiently by frequently occurring oncogenic Ras mutants, to yield guanosine 5'-diphosphate-bound inactive Ras and DABP-Pi. The reaction is independent of the presence of Gln61 and is most dramatically enhanced with Gly12 mutants. Thus, the defective GTPase reaction of the oncogenic Ras mutants can be rescued by using DABP-GTP instead of GTP, arguing that the GTPase switch of Ras is not irreversibly damaged. An exocyclic aromatic amino group of DABP-GTP is critical for the reaction and bypasses the putative rate-limiting step of the intrinsic Ras GTPase reaction. The crystal structures of Ras-bound DABP-beta,gamma-imido-GTP show a disordered switch I and identify the Gly12/Gly13 region as the hydrophobic patch to accommodate the DABP-moiety. The biochemical and structural studies help to define the requirements for the design of anti-Ras drugs aimed at the blocked GTPase reaction.
The crystal structure of the complex between a G protein alpha subunit (Gi alpha 1) and its GTPase-activating protein (RGS4) demonstrated that RGS4 acts predominantly by stabilization of the transition state for GTP hydrolysis [Tesmer, J. J., et al. (1997) Cell 89, 251]. However, attention was called to a conserved Asn residue (Asn128) that could play a catalytic role by interacting, directly or indirectly, with the hydrolytic water molecule. We have analyzed the effects of several disparate substitutions for Asn128 on the GAP activity of RGS4 toward four G alpha substrates (Go, Gi, Gq, and Gz) using two assay formats. The results substantiate the importance of this residue but indicate that it is largely involved in substrate binding and that its function may vary with different G alpha targets. Various mutations decreased the apparent affinity of RGS4 for substrate G alpha proteins by several orders of magnitude, but had variable and modest effects on maximal rates of GTP hydrolysis when tested with different G alpha subunits. One mutation, N128F, that differentially decreased the GAP activity toward G alpha i compared with that toward G alpha q could be partially suppressed by mutation of the nearby residue in G alpha i to that found in G alpha q (K180P). Detection of GAP activities of the mutants was enhanced in sensitivity up to 100-fold by assay at steady state in proteoliposomes that contain heterotrimeric G protein and receptor.
Substrate-assisted catalysis (SAC) is the process by which a functional group in a substrate contributes to catalysis by an enzyme. SAC has been demonstrated for representatives of three major enzyme classes: serine proteases, GTPases, and type II restriction endonucleases, as well as lysozyme and hexose-1-phosphate uridylyltransferase. Moreover, structure-based predictions of SAC have been made for many additional enzymes. Examples of SAC include both naturally occurring enzymes such as type II restriction endonucleases as well as engineered enzymes including serine proteases. In the latter case, a functional group from a substrate can substitute for a catalytic residue replaced by site-directed mutagenesis. From a protein engineering perspective, SAC provides a strategy for drastically changing enzyme substrate specificity or even the reaction catalyzed. From a biological viewpoint, SAC contributes significantly to the activity of some enzymes and may represent a functional intermediate in the evolution of catalysis. This review focuses on advances in engineering enzyme specificity and activity by SAC, together with the biological significance of this phenomenon.
Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes Ras oncogenes in human cancer: a review The expanding spectrum of G protein diseases
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How does GAP catalyze the GTPase reaction of Ras? A computer simulation study Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site
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GTPase-activating proteins: helping hands to complement an active site Signaling mechanistics: aluminum fluoride for molecule of the year
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Catecholaminestimulated GTPase activity in turkey erythrocyte membranes
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Fersht, A. (1985) Enzyme Structure and
Mechanism, W.H. Freeman
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membranes. Biochim. Biophys. Acta 452,
538–551