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Structural analysis of human liver glyceraldehyde-3-phosphate dehydrogenase
Abstract
The crystal structure of human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been determined. This structure represents the first moderate-resolution (2.5 A) and crystallographically refined (Rfree = 22.9%) human GAPDH structure. The liver GAPDH structure consists of a homotetramer, each subunit of which is bound to a nicotinamide adenine dinucleotide (NAD+) molecule. The GAPDH enzyme has glycolytic and non-glycolytic functions, both of which are of chemotherapeutic interest. The availability of a high-quality human GAPDH structure is a necessity for structure-based drug design. In this study, structural differences between human liver and skeletal muscle GAPDHs are reported in order to understand how these two enzymes might respond to anti-trypanosomatid GAPDH inhibitors.
... GAPDH enzymes isolated from various sources do not differ markedly with respect to their sedimentation coefficients [7] or their amino acid composition [7][8][9]. Because of the extensive homology in its amino acid sequence among different sources, GAPDH is also used for studying evolutionary relationships [10]. ...
... The enzyme with 144 kDa consists of 4 identical subunits, as indicated by the letters O, P, Q, and R in (Fig. 1B). Each 330-amino acid subunit has 2 domains, 1 of which binds NAD + (NAD +binding domain) [8,10,13,14]. The NAD + -binding domain comprises amino acids 1-148 [10]. ...
... Each 330-amino acid subunit has 2 domains, 1 of which binds NAD + (NAD +binding domain) [8,10,13,14]. The NAD + -binding domain comprises amino acids 1-148 [10]. The other is the catalytic domain (residues 149-329), containing a cysteine residue (Cys-149) that is involved in the enzyme-coenzyme chargetransfer interactions [10,15,16]. ...
D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reversible oxidation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bis-phospho-D-glycerate, and as such participates in the glycolytic conversion of glucose to pyruvic acid in most living organisms. The glycolytic pathway plays an important role in tumor cells, but it is not clear whether the enzyme kinetics of GAPDH or their response to inhibitors, substrates, and cofactors differ between tumor cells and normal cells. To obtain a tumor-derived GAPDH sample, HeLa cells were chosen on the basis of their homogeneous differentiation pattern and ease of harvesting. We carried out experiments to investigate whether the enzyme kinetics of purified GAPDH from HeLa cells were altered in the presence of reagents containing sulfhydryl groups, divalent metal ions, and cellular metabolites such as nucleotides and coenzymes. The kinetic data were compared with data for GAPDH from normal tissue. GAPDH from HeLa cells was activated by 2-mercaptoethanol and dithioerythritol. The maximum activation was obtained at a 1 mM concentration of each reducing agent. Cupric and mercuric ions (1 mM), as well as p-chloro and phydroxymercuribenzoate (10 mM), fully inhibited enzymatic activity. Among the nucleotides tested, 3¢–5¢-cyclic AMP (cAMP) was the most effective inhibitor at 30 mM concentration, with a relative activity of 22.79 (±1.76), which was significantly different (p £ 0.05) from that of the control, which had 100% activity in the absence of adenine nucleotide. Enzyme inhibition by adenine nucleotides appeared to be via competition with NAD+. The apparent inhibition constants (Ki) for ADP, 5¢-AMP, and cAMP were 2.1 mM, 1.0 mM, and 0.6 mM, respectively. GAPDH from HeLa cells was inactivated when incubated in the presence of G3P or NADH at 37°C, and in both cases the presence of 2-mercaptoethanol protected the enzyme against inhibition. The presence of EDTA did not affect the inactivation of the enzyme by NADH, which suggested that the inactivation of HeLa GAPDH by NADH is not related to the presence of heavy metal ions. Our kinetic analysis showed that although the GAPDH of HeLa cells has a lower specific activity and stability compared with GAPDH from normal tissue, its kinetic characteristics were similar, reinforcing the key role of this enzyme in the metabolism of tumor cells.
... As the nicotinamide end of NAD + faces the interior of the GAPDH tetramer, a cleft opens, revealing a spacious active-site pocket ( Fig. 1) [2]. The catalytic domain, on the other hand, consists of twisted, eight-stranded parallel β-sheets connected by α-helices on one side, while the other side of this β-sheet forms extensive contacts with the β-sheet of an adjacent subunit ( Fig. 1) [2,69,70]. In addition, the amino acid Val-240 (human analog) of each subunit falls in a disallowed region of Ramachandran plots, which is a characteristic of all GAPDH enzymes [70][71][72][73][74]. ...
... The hemiacetal intermediate is then oxidized to a high energy thioester, by hydride transfer to NAD + , possibly facilitated by His-179 in the catalytic domain [75,76]. This high energy thioester is then attacked by nucleophilic, inorganic phosphate (P i ) to form BPG (Fig. 4) [68,69,77]. Mutational studies in Bacillus stearothermophilus show replacement of Cys-149 (human analog Cys-152) with a Ser thiol significantly reduces GAPDH activity, while substitution with Ala completely inactivates the enzyme [77,78]. ...
... Analysis of such functions after proteolysis and competitive binding using NAD + and polynucleotides indicates that the majority of GAPDH functional diversity is a direct result of the many different interactions that can occur within the NAD + -binding domain [1,26,[82][83][84][85]. For example, the NAD + -binding domain Rossmann fold, essential to the dehydrogenase activity of GAPDH [2,69,70], is also important in the catalysis of aminoacyl tRNA synthesis by GAPDH [86], and may comprise the GAPDH nucleotide binding site for tubulin [87]. Moreover, GAPDH can act as a nuclear tRNA transport protein, whose activity is inhibited by NAD + in a competitive manner [1,88]. ...
Recently, the oxidoreductase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has become a subject of interest as more and more studies reveal a surfeit of diverse GAPDH functions, extending beyond traditional aerobic metabolism of glucose. As a result of multiple isoforms and cellular locales, GAPDH is able to come in contact with a variety of small molecules, proteins, membranes, etc., that play important roles in normal and pathologic cell function. Specifically, GAPDH has been shown to interact with neurodegenerative disease-associated proteins, including the amyloid-beta protein precursor (AbetaPP). Studies from our laboratory have shown significant inhibition of GAPDH dehydrogenase activity in Alzheimer's disease (AD) brain due to oxidative modification. Although oxidative stress and damage is a common phenomenon in the AD brain, it would seem that inhibition of glycolytic enzyme activity is merely one avenue in which AD pathology affects neuronal cell development and survival, as oxidative modification can also impart a toxic gain-of-function to many proteins, including GAPDH. In this review, we examine the many functions of GAPDH with respect to AD brain; in particular, the apparent role(s) of GAPDH in AD-related apoptotic cell death is emphasized.
... Among phosphorylation, acetylation, ubiquitylation and redox-PTMs, GAPDH has been reported to be succinylated at various surface lysines 6 and glutarylated at K194 5 , a residue close to the active site. In the cytosol, GAPDH forms a homotetramer that is stabilized by several hydrogen bonds between the individual subunits ( Fig. 3a) 39 . Each monomer consists of a C-terminal cofactor-binding domain and an N-terminal catalytic domain harbouring the catalytic residues C152 and H179. ...
... Interacting S-loop regions of two adjacent monomers (blue and black) are coloured in yellow and dark grey, respectively. K194 is shown in red, and NAD + in pink (Protein Data Bank, 1znq)39 . Right: Structure of the GAPDH monomer highlighting the cofactor-binding domain, the catalytic domain and S-loop. ...
Post-translational modifications (PTMs) dynamically regulate cellular processes. Lysine undergoes a range of acylations, including malonylation, succinylation (SucK) and glutarylation (GluK). These PTMs increase the size of the lysine side chain and reverse its charge from +1 to −1 under physiological conditions, probably impacting protein structure and function. To understand the functional roles of these PTMs, homogeneously modified proteins are required for biochemical studies. While the site-specific encoding of PTMs and their mimics via genetic code expansion has facilitated the characterization of the functional roles of many PTMs, negatively charged lysine acylations have defied this approach. Here we describe site-specific incorporation of SucK and GluK into proteins via temporarily masking their negative charge through thioester derivatives. We prepare succinylated and glutarylated bacterial and mammalian target proteins, including non-refoldable multidomain proteins. This allows us to study how succinylation and glutarylation impact enzymatic activity of metabolic enzymes and regulate protein–DNA and protein–protein interactions in biological processes from replication to ubiquitin signalling.
... However, both N-and C-terminal residues contribute to the binding. The NAD + -binding site has been documented, where residues R13, I14, D35, R80, S122, and N316 are involved in forming this coenzyme-binding site (Ismail & Park, 2005;Jenkins & Tanner, 2006) (Fig 4D). Unfortunately, we did not see nucleotides occupying this NAD + -binding site in our cryo-EM structure. ...
... The substrate-binding site is located at the interface between the N-terminal and C-terminal domains. It has been reported that residues S151, C152, T153, T182, T211, G212, and R234 are involved in forming the catalytic site (Ismail & Park, 2005). Again, our cryo-EM structure suggests that this catalytic site is unoccupied (Fig 4E). ...
We recently developed a “Build and Retrieve” cryo-electron microscopy (cryo-EM) methodology, which is capable of simultaneously producing near-atomic resolution cryo-EM maps for several individual proteins from a heterogeneous, multiprotein sample. Here we report the use of “Build and Retrieve” to define the composition of a raw human brain microsomal lysate. From this sample, we simultaneously identify and solve cryo-EM structures of five different brain enzymes whose functions affect neurotransmitter recycling, iron metabolism, glycolysis, axonal development, energy homeostasis, and retinoic acid biosynthesis. Interestingly, malfunction of these important proteins has been directly linked to several neurodegenerative disorders, such as Alzheimer’s, Huntington’s, and Parkinson’s diseases. Our work underscores the importance of cryo-EM in facilitating tissue and organ proteomics at the atomic level.
... GAPDH is a 335 amino acid-long polypeptide which is highly conserved across the phylogenetic scale. It contains two major domains, the NAD + -binding domain which forms the main chain, and the catalytic glyceraldehyde-3-phosphate (G3P)-binding domain which forms the Cterminal helix ( Fig. 1) (Ismail and Park, 2005;Jenkins and Tanner, 2006). The sequence and structure of the N-terminal NAD binding domain (amino acids 1-150) are similar to those found in other dehydrogenases (for example lactate or alcohol). ...
... This area is responsible for binding dinucleotides (Cowan-Jacob et al., 2003;Rodacka, 2013). The catalytic domain of GAPDH (amino acid 151-335) is complexed with eight parallel twisted fragments of β-sheet structures, connected on, one side by short α-helix sections; on the other hand, the structures of β-sheets expand extensively with the same areas of the neighbouring catalytic domain subunit (Skarzynski et al., 1987;Yun et al., 2000;Ismail and Park, 2005;Sirover, 2014). This area is responsible for the specific catalytic activities of the reaction sequence, and substrate specificity. ...
A number of independent studies have shown the contribution of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the pathogenesis of several neurodegenerative disorders. Indeed, GAPDH aggregates have been found in many post-mortem samples of brains of patients diagnosed with Alzheimer’s and Parkinson disease. Currently, it is accepted that GAPDH-mediated cell death pathways in the neurodegenerative processes are associated with apoptosis caused by GAPDH nuclear translocation and excessive aggregation under oxidative stress conditions. Also the role of GAPDH in neurodegenerative diseases is linked to it directly binding to specific amyloidogenic proteins and petides such as β-amyloid precursor protein, β-amyloid peptide and tau protein in Alzheimer’s disease, huntingtin in Huntington’s disease and α-synuclein in Parkinson disease. One of the latest studies indicated that GAPDH aggregates significantly accelerate amyloidogenesis of the β-amyloid peptide, which implies that aggregates of GAPDH may act as a specific aggregation “seed” in vitro.
Previous detailed studies revealed that the active-site cysteine (Cys152) of GAPDH plays an essential role in the oxidative stress-induced aggregation of GAPDH associated with cell death. Furthermore, oxidative modification of this cysteine residue initiates the translocation of the enzyme to the nucleus, subsequently leading to apoptosis. The crystallographic structure of GAPDH shows that the Cys152 residue is located close to the surface of the molecule in a hydrophilic environment, which means that it can react with low molecular weight compounds such as hydroxynonenal or piceatannol. Therefore, it is highly possible that GAPDH may serve as a target for small molecule compounds with the potential to slow down or prevent the progression of neurodegenerative disorders.
Recently appearing new evidence has highlighted the significance of low molecular weight compounds in counteracting the oxidation of GAPDH and consequently its aggregation and other unfavourable pathological processes. Hence, this review aims to present all recent findings concerning molecules that are able to interact with GAPDH and counteract its aggregation and translocation to the nucleus.
... The GAPDH tetramer contains four possible NAD + -binding sites. Crystal structures with 2 (Cowan- Jacob et al. 2003), 3 (Jenkins and Tanner 2006;White et al. 2015), or 4 (Baker et al. 2014Chaikuad et al. 2011;Ismail and Park 2005) NAD + molecules have been determined, but no crystal structure of the apo human GAPDH enzyme is available. In our hands, we find that purified GAPDH almost always contains NAD + even in the absence of added cofactor during bacterial growth or protein purification. ...
... In addition, of the S-loop from the subunit across the Q interface also contribute to the cofactor-binding site (Fig. 15.3a). This loop usually adopts a "closed" conformation close to the adjacent subunit (White et al. 2015), but has also been observed in an "open" conformation further from the adjacent subunit (Ismail and Park 2005). The significance of this difference is not clear, but could be due to crystallographic artifacts. ...
Aside from its well-established role in glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been shown to possess many key functions in cells. These functions are regulated by protein oligomerization
, biomolecular interactions, post-translational modifications
, and variations in subcellular localization
. Several GAPDH functions and regulatory mechanisms overlap with one another and converge around its role in intermediary metabolism. Several structural determinants of the protein dictate its function and regulation. GAPDH is ubiquitously expressed and is found in all domains of life. GAPDH has been implicated in many diseases, including those of pathogenic, cardiovascular, degenerative, diabetic, and tumorigenic origins. Understanding the mechanisms by which GAPDH can switch between its functions and how these functions are regulated can provide insights into ways the protein can be modulated for therapeutic outcomes.
... Therefore, V5-tagged WT or M6 GAPDH expression vectors were prepared and cotransfected into HEK293 cells with pNL-CH to investigate whether the viruses produced from M6-GAPDH-expressing cells showed suppressed GAPDH packaging and restored tRNA Lys3 packaging efficiency. We first performed coimmunoprecipitation assay to validate whether endogenous GAPDH and exogenous V5-tagged GAPDH retained oligomatic formation, because GAPDH exists primarily as a homotetramer in the cytoplasm [17]. The assay showed that both V5-tagged WT and M6 GAPDH retained oligomatic formation with endogenous GAPDH in HIV-1 producer cells (Fig. 4C), ...
... The GAPDH mutagenesis assay indicated that Asp 256 , Lys 260 , Lys 263 and Glu 267 of GAPDH are important residues in the interaction of GAPDH with MA and CA-NTD. Importantly, the crystal structure of GAPDH tetramer (PDB ID: 1ZNQ) [17] shows that all of these amino acids in helix 10 are exposed on four each of monomer GAPDH, suggesting that two of such studies demonstrated that the tRNA Lys3 /LysRS complex interacts with the Pr55 gag /p160 gag-pol /viral genome RNA complex and is efficiently packaged into virions [4,5,9,20,21]. Javanbakht et al. [5] demonstrated that the domains critical for the Pr55 gag -LysRS interaction are mapped to include the dimerization domains of both LysRS and CA. ...
Human immunodeficiency virus type-1 (HIV-1) requires the packaging of human tRNALys3 as a primer for effective viral reverse transcription. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) suppresses the packaging efficiency of tRNALys3. Although the binding of GAPDH to Pr55
gag
is important for the suppression mechanism, it remains unclear which domain of GAPDH is responsible for the interaction with Pr55
gag
. In this study, we show that Asp256, Lys260, Lys263 and Glu267 of GAPDH are important for the suppression of tRNALys3 packaging. Yeast two-hybrid analysis demonstrated that the C-terminal domain of GAPDH (151-335) interacts with both the matrix region (MA; 1-132) and capsid N-terminal domain (CA-NTD; 133-282). The D256R, K263E or E267R mutation of GAPDH led to the loss of the ability to bind to wild-type (WT) MA, and the D256R/K260E double mutation of GAPDH resulted in the loss of detectable binding activity to WT CA-NTD. In contrast, R58E, Q59A or Q63A of MA, and E76R or R82E of CA-NTD abrogated the interaction with the C-terminal domain of GAPDH. Multiple-substituted GAPDH mutant (D256R/K260E/K263E/E267R) retained the oligomeric formation with WT GAPDH in HIV-1 producing cells, but the incorporation level of the hetero-oligomer was decreased in viral particles. Furthermore, the viruses produced from cells expressing the D256R/K260E/K263E/E267R mutant restored tRNALys3 packaging efficiency because the mutant exerted a dominant negative effect by preventing WT GAPDH from binding to MA and CA-NTD and improved the reverse transcription. These findings indicate that the amino acids Asp256, Lys260, Lys263 and Glu267 of GAPDH is essential for the mechanism of tRNALys3-packaging suppression and the D256R/K260E/K263E/E267R mutant of GAPDH acts in a dominant negative manner to suppress tRNALys3 packaging.
... In both cases, the consensus tetramer of the GAPDH family is formed by crystallographic symmetry that recapitulates the biological tetramer. As reported for multiple GAPDH and GAPDHS structures (Cowan-Jacob et al., 2003;Ismail and Park, 2005;Frayne et al., 2009), the active site cysteines (C224 in human GAPDHS, C256 in mouse GAPDHS) in our structures were oxidized to the sulfinic acid form, with clear electron density for two oxygens attached to the cysteine sulfur. ...
... This is an intersubunit cleft across the R-axis that is adjacent to the adenosine ribose of NAD + in the neighboring subunit. 'Open' (PDB 1ZNQ; Ismail and Park, 2005) and 'closed' (PDB 1U8F; Jenkins and Tanner, 2006) conformations of the S-loop have been reported in human GAPDH structures, although the distance between residues closest to NAD + (S-loop P191 and F37 in the Figure 10 Inhibition of mouse sperm lactate production by T0501_7749. Mouse sperm were incubated for 2 h under the same conditions as in Fig. 9A, except that lactate and pyruvate were omitted from the HTF medium. ...
STUDY HYPOTHESIS Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the
somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS
inhibitors for contraceptive development.
... The amino acid residues of NS1 involved in the surface contact with the GAPDH structure are Cys4, Val5, Val6, Asn10, Lys11, Leu13, Lys14, Cys15, and Trp28 of one monomer and Gly3, Cys4, Val5, Glu12, Leu13, Lys14, Cys15, Gly161, Val162, Phe163, and Thr164 of the other monomer ( Fig. 4C and D). As GAPDH has two important catalytic residues, cysteine 152 (Cys152) and histidine 179 (His179) (38), these were also highlighted (in green) to evaluate whether the interaction may affect this region. It was observed that NS1 interacted on the opposite side of the GAPDH catalytic site (Fig. 4A and B), suggesting that if NS1 affects GAPDH activity, it is not because the catalytic site is blocked. ...
... The reaction catalyzed by GAPDH starts with the production of a thiohemiacetal intermediate at the Cys152 residue, followed by the formation of a high-energy thioester that is then attacked by P i to generate BPG. After thioester formation, a hydride is transferred to an NAD ϩ molecule with the aid of the His179 residue, which acts as a base catalyst (38). The molecular docking results suggest that the NS1-GAPDH interaction occurs through hydrophobic protrusions in the NS1 -roll domain and the hydrophobic residues of GAPDH, located on the opposite side of the catalytic site. ...
Importance:
Dengue represents a serious public health problem worldwide and is caused by the infection of dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the intracellular and extracellular milieu. Despite the fact that NS1 has been commonly associated with DENV pathogenesis, it plays a pivotal, but unknown role in the replication process. In an effort to understand the role of intracellular NS1, we demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with NS1. Our results indicated that NS1 increases the glycolytic activity of GAPDH in vitro. Interestingly, the GAPDH activity was increased during DENV infection, and NS1 expression alone was sufficient to enhance intracellular GAPDH activity in BHK-21 cells. Overall, our findings suggest that NS1 is an important modulator of cellular energy metabolism by increasing glycolytic flux.
... GAPDH C156S was labeled with mixed thiol-reactive dyes, Alexa 546-and 647-C5-maleimide. The apparent mean interfluor distance for GAPDH by itself was calculated to be 51.6 Å and increased to 58.0 Å by addition of S100A8 ( Figure S2), indicative of a substantial conformational shift, consistent with S-nitrosylation of tetrameric GAPDH as the X-ray structure (Ismail and Park, 2005) indicates the distance between the Cys-Cys pair in the monomer is 21 Å, whereas the maximum distance in the tetramer is 64.8 Å (Ismail and Park, 2005). ...
... GAPDH C156S was labeled with mixed thiol-reactive dyes, Alexa 546-and 647-C5-maleimide. The apparent mean interfluor distance for GAPDH by itself was calculated to be 51.6 Å and increased to 58.0 Å by addition of S100A8 ( Figure S2), indicative of a substantial conformational shift, consistent with S-nitrosylation of tetrameric GAPDH as the X-ray structure (Ismail and Park, 2005) indicates the distance between the Cys-Cys pair in the monomer is 21 Å, whereas the maximum distance in the tetramer is 64.8 Å (Ismail and Park, 2005). ...
S-nitrosylation is a ubiquitous protein modification emerging as a principal mechanism of nitric oxide (NO)-mediated signal transduction and cell function. S-nitrosylases can use NO synthase (NOS)-derived NO to modify selected cysteines in target proteins. Despite proteomic identification of over a thousand S-nitrosylated proteins, few S-nitrosylases have been identified. Moreover, mechanisms underlying site-selective S-nitrosylation and the potential role of specific sequence motifs remain largely unknown. Here, we describe a stimulus-inducible, heterotrimeric S-nitrosylase complex consisting of inducible NOS (iNOS), S100A8, and S100A9. S100A9 exhibits transnitrosylase activity, shuttling NO from iNOS to the target protein, whereas S100A8 and S100A9 coordinately direct site selection. A family of proteins S-nitrosylated by iNOS-S100A8/A9 were revealed by proteomic analysis. A conserved I/L-X-C-X2-D/E motif was necessary and sufficient for iNOS-S100A8/A9-mediated S-nitrosylation. These results reveal an elusive parallel between protein S-nitrosylation and phosphorylation, namely, stimulus-dependent posttranslational modification of selected targets by primary sequence motif recognition.
... domenę wiążącą koenzym NAD + (reszty aminokwasowe 1-151 oraz 315-335) i C-terminalną domenę katalityczną (reszty aminokwasowe 152-314) [13,36,38]. ...
... Obszar ten jest odpowiedzialny za przebieg reakcji chemicznej oraz swoistość substratową. Złożony jest z ośmiu równolegle skręconych fragmentów o strukturze β-kartek, połączonych z jednej strony krótkimi odcinkami α-helisy, z drugiej strony struktury β rozlegle oddziałują z tymi samymi obszarami domeny katalitycznej sąsiedniej podjednostki [36,38]. ...
... An initial molecular model of a FhGAPDH monomer was generated using Phyre2 [44]. Four of these monomeric structures were superimposed onto the subunits of human liver GAPDH (PDB: 1ZNQ [45]) using PyMol (www.pymol.org) to generate an initial model of tetrameric FhGAPDH. This initial model was energy minimised using YASARA [46] to generate the final model which is provided as supplementary data to this paper. ...
... FhGAPDH monomer was generated using Phyre2 [44]. Four of these monomeric structures were superimposed onto the subunits of human liver GAPDH (PDB: 1ZNQ [45]) using PyMol (www.pymol.org) to generate an initial model of tetrameric FhGAPDH. This initial model was energy minimised using YASARA [46] to generate the final model which is provided as supplementary data to this paper. ...
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses one of the two steps in glycolysis which generate the reduced coenzyme NADH. This reaction precedes the two ATP generating steps. Thus, inhibition of GAPDH will lead to substantially reduced energy generation. Consequently, there has been considerable interest in developing GAPDH inhibitors as anti-cancer and anti-parasitic agents. Here, we describe the biochemical characterisation of GAPDH from the common liver fluke Fasciola hepatica (FhGAPDH). The primary sequence of FhGAPDH is similar to that from other trematodes and the predicted structure shows high similarity to those from other animals including the mammalian hosts. FhGAPDH lacks a binding pocket which has been exploited in the design of novel antitrypanosomal compounds. The protein can be expressed in, and purified from Escherichia coli; the recombinant protein was active and showed no cooperativity towards glyceraldehyde 3-phosphate as a substrate. In the absence of ligands, FhGAPDH was a mixture of homodimers and tetramers, as judged by protein-protein crosslinking and analytical gel filtration. The addition of either NAD(+) or glyceraldehyde 3-phosphate shifted this equilibrium towards a compact dimer. Thermal scanning fluorimetry demonstrated that this form was considerably more stable than the unliganded one. These responses to ligand binding differ from those seen in mammalian enzymes. These differences could be exploited in the discovery of reagents which selectively disrupt the function of FhGAPDH.
... domenę wiążącą koenzym NAD + (reszty aminokwasowe 1-151 oraz 315-335) i C-terminalną domenę katalityczną (reszty aminokwasowe 152-314) [13,36,38]. ...
... Obszar ten jest odpowiedzialny za przebieg reakcji chemicznej oraz swoistość substratową. Złożony jest z ośmiu równolegle skręconych fragmentów o strukturze β-kartek, połączonych z jednej strony krótkimi odcinkami α-helisy, z drugiej strony struktury β rozlegle oddziałują z tymi samymi obszarami domeny katalitycznej sąsiedniej podjednostki [36,38]. ...
For a long time glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered a classical glycolytic protein of little interest. It was also used as a model protein for analysis of protein structure and enzyme mechanisms. However, recent evidence demonstrates that GAPDH from mammalian cells displays a number of diverse activities unrelated to its glycolytic function. This enzyme is an example of moonlighting protein. Dehydrogenase participates in membrane fusion, microtubule assembly, vesicular transport, and the maintenance of DNA integrity. New and novel studies indicate that enzyme is directly involved in transcriptional, posttranscriptional gene regulation, and the maintenance of chromatin structure. Furthermore, other studies also indicate a role of GAPDH in apoptosis, and age-related neurodegenerative disease e.g. Alzheimer's, Huntington's and Parkinson's diseases. This work describes the structure and localization of GAPDH in cells as well as the latest discoveries on the multifunctional properties of the enzyme.
... Bovine GAPDH assembles as a tetrameric oligomer ( Figures 1A and 1B), which is in good agreement with crystal structures of the human, 18 bovine, 19 lobster, 20 and Bacillus stearothermophilus 21 GAPDH enzymes. Superimposition of the cryo-EM structure of bovine GAPDH to its X-ray structure (PDB: 4O59) 19 gives rise to a root-mean-square deviation (RMSD) of 0.49 Å (for 328 Cα atoms). ...
The use of an integrated systems biology approach to investigate tissues and organs has been thought to be impracticable in the field of structural biology, where the techniques mainly focus on determining the structure of a particular biomacromolecule of interest. Here, we report the use of cryoelectron microscopy (cryo-EM) to define the composition of a raw bovine retinal pigment epithelium (RPE) lysate. From this sample, we simultaneously identify and solve cryo-EM structures of seven different RPE enzymes whose functions affect neurotransmitter recycling, iron metabolism, gluconeogenesis, glycolysis, axonal development, and energy homeostasis. Interestingly, dysfunction of these important proteins has been directly linked to several neurodegenerative disorders, including Huntington’s disease, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Alzheimer’s disease, and schizophrenia. Our work underscores the importance of cryo-EM in facilitating tissue and organ proteomics at the atomic level.
... Glyceraldehyde 3-phosphate dehydrogenase (abbreviated as PfGAPDH for Plasmodium falciparum) is a key glycolytic enzyme that catalyzes the conversion of Glyceraldehyde 3-phosphate (G3P) into 1,3-bisphosphate glycerate (1,, with the concomitant reduction of nicotinamide adenine dinucleotide (NAD + ) to NADH [8]. This is the 6th step in the glycolytic breakdown of glucose, an important pathway of energy and carbon molecule supply which takes place in the cytosol of eukaryotic cells [9]. ...
To search for leads against malaria, compounds from the Cameroon Natural Product Library (CANAPL), were screened by molecular docking studies against the enzyme target Glyceraldehyde-3-phosphate dehydrogenase (P f GAPDH) for Plasmodium falciparum . Amongst these docking hits were the lupeol cinnamate (1) and oleanane cinnamate (2). Triterpenoids with structural similarities to (1) and (2) were isolated from Baillonella toxisperma (Pierre); olean-12-en-3β-hexadecanoate (3), 3-β-trans cinnamoyloxylup-20(29)-ene (4), oleanoic acid benzoate (5), 3β-amyrin (6), taraxerol (7), betulonic acid (8), β-sitosterol (9), betulinic acid (10), 3β-(trans-p-Coumaroyl)oxylup-20(29)-en-28-oïc acid (11), and screened alongside Betuline (12) and oleanolic acid (13). The favorable compounds 8 and 11 with 100% growth inhibition on Plasmodium falciparum 3D7 strains together with compounds 4, 10 and 12 were structurally optimized to afford new lupane derivatives with the privilege α-β unsaturated carbonyl medicinal scaffolds; betulonic acid acryl aldehyde (14 ) , betulin acryl aldehyde (15), 3β-( trans-p -Coumaroyl)oxylup-20(29)-en-28-oïc acid acryl aldehyde (16), betulinic acid acryl aldehyde (18) and 3-β- trans cinnamoyloxylup-20(29)-ene acryl aldehyde (19) exhibiting more potent anti-malarial activities with IC 50 values of 0.703 µM, 2.15 µM, 1.28 µM, and 3.79 µM for compounds 14, 15, 16 and 18 respectively. The modified compounds with the privilege α, β-unsaturated carbonyl medicinal scaffolds could be further optimized into potent antimalarial agents.
... Comparison with the structure of human liver GAPDH (HsGAPDH) (PDB ID 1ZNQ) (Ismail and Park, 2005) is informative because structural and chemical features that are distinctive between the GAPDH of bacterial pathogens and HsGAPDH can be exploited for the discovery of selective inhibitors. Selective inhibitors avoiding the first pass metabolism in the liver have in principle two advantages: (1) their concentrations in systemic circulation will probably be greater, and (2) less adverse effects by not interfering with liver HsGAPDH's physiological function. ...
The ubiquitous and highly abundant glycolytic enzyme D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is pivotal for the energy and carbon metabolism of most organisms, including human pathogenic bacteria. For bacteria that depend mostly on glycolysis for survival, GAPDH is an attractive target for inhibitor discovery. The availability of high-resolution structures of GAPDH from various pathogenic bacteria is central to the discovery of new antibacterial compounds. We have determined the X-ray crystal structures of two new GAPDH enzymes from Gram-positive bacterial pathogens, Streptococcus pyogenes and Clostridium perfringens. These two structures, and the recent structure of Atopobium vaginae GAPDH, reveal details in the active site that can be exploited for the design of novel inhibitors based on naturally occurring molecules. Two such molecules, anacardic acid and curcumin, have been found to counter bacterial infection in clinical settings, although the cellular targets responsible for their antimicrobial properties remain unknown. We show that both anacardic acid and curcumin inhibit GAPDH from two bacterial pathogens through uncompetitive and non-competitive mechanisms, suggesting GAPDH as a relevant pharmaceutical target for antibacterial development. Inhibition of GAPDH by anacardic acid and curcumin seems to be unrelated to the immune evasion function of pathogenic bacterial GAPDH, since neither natural compound interfere with binding to the human C5a anaphylatoxin.
... AdOx treatment led to a 30% decrease in the Km(NAD + ), whereas the Km(G3P) remained unchanged ( Figure 2B). The crystal structure of GAPDH indicates that R234 is located in the catalytic center and that R234 potentially modulates substrate binding; that is, the affinity of GAPDH for G3P and NAD + ( Figure S1B; Ismail and Park, 2005;Jenkins and Tanner, 2006). We hypothesized that R234 methylation might inhibit GAPDH activity by regulating the affinity of GAPDH for G3P and/or NAD + . ...
Increased aerobic glycolysis is a hallmark of cancer metabolism. How cancer cells coordinate glucose metabolism with extracellular glucose levels remains largely unknown. Here, we report that coactivator-associated arginine methyltransferase 1 (CARM1 or PRMT4) signals glucose availability to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and suppresses glycolysis in liver cancer cells. CARM1 methylates GAPDH at arginine 234 (R234), inhibiting its catalytic activity. Glucose starvation leads to CARM1 upregulation, further inducing R234 hypermethylation and GAPDH inhibition. The re-expression of wild-type GAPDH, but not of its methylation-mimetic mutant, sustains glycolytic levels. CARM1 inhibition increases glycolytic flux and glycolysis. R234 methylation delays tumor cell proliferation in vitro and in vivo. Compared with normal tissues, R234 is hypomethylated in malignant clinical hepatocellular carcinoma samples. Notably, R234 methylation positively correlates with CARM1 expression in these liver cancer samples. Our findings thus reveal that CARM1-mediated GAPDH methylation is a key regulatory mechanism of glucose metabolism in liver cancer. GAPDH is a critical enzyme in glycolysis. Zhong et al. find that CARM1 methylates GAPDH at R234 and inhibits its activity in an AMPK-dependent manner. R234 methylation inhibits glycolysis and proliferation of liver cancer cell lines. In hepatocellular carcinoma patient samples, GADPH R234 is hypomethylated, and there is a positive correlation between CARM1 levels and R234 methylation.
... The second protein in the glycolytic pathway is glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This enzyme catalyzes the sixth step of glycolysis, during which a molecule of GA3-P is converted to 1,3-bisphosphoglycerate with production of NADH (130). Although GAPDH also possesses other functions, including DNA/RNA binding, gene transcription, and protein interaction (34), its role in the glycolytic process is unquestionable. ...
Alzheimer disease (AD), a multifactorial neurodegenerative disorder that represents one of the most disabling conditions in the aged population, shares many features in common with systemic insulin resistance diseases, including reduced insulin-stimulated growth and survival signaling, increased oxidative stress, pro-inflammatory cytokine activation, mitochondrial dysfunction, impaired energy metabolism and altered protein homeostasis. Reduced glucose utilization and energy metabolism in AD brain is associated with the accumulation of: 1) Aβ peptide and hyperphosphorylated tau; 2) increased oxidative stress; 3) unfolded/misfolded proteins. mTOR, aberrantly activated in AD from its earliest stages, plays a key role in AD neurodegeneration by both inhibiting insulin signalling as a negative feedback mechanism and regulating protein homeostasis (synthesis/clearance). Employing the techniques of redox proteomics pioneered in our laboratory led to the identification of oxidatively modified brain proteins in AD involved in concomitant and mutual alterations of energy metabolism, mTOR signaling, and protein homeostasis. These proteins form a self-sustaining triangle of harmful events that trigger the degeneration and death of neurons and the development and progression of AD. Moreover, the altered crosstalk among the components of this “triangle of death”, beyond altering the redox homeostasis of neurons, is further exacerbated by increased levels of oxidative stress that target and impair key components of the pathways involved, thereby revealing the crucial role of oxidative stress in fueling this aberrant vicious cycle. This triangle of death may represent promising therapeutic targets to slow, delay, or prevent progression of AD.
... GAPDH functions as a tetramer, and examination of the 3D crystal structure indicated that the C terminus of each GAPDH subunit is located on the exterior surface of the tetramer (Ismail and Park, 2005), suggesting that additional amino acids encoded by the T2A sequence should not interfere with enzymatic activity. However, examination of GAPDH protein using western blot analysis indicated that modified alleles that include an internal ribosome entry site (IRES)-selectable marker cassette were expressed at lower levels than the wild-type GAPDH allele. ...
... GAPDH functions as a tetramer, and examination of the 3D crystal structure indicated that the C terminus of each GAPDH subunit is located on the exterior surface of the tetramer (Ismail and Park, 2005), suggesting that additional amino acids encoded by the T2A sequence should not interfere with enzymatic activity. However, examination of GAPDH protein using western blot analysis indicated that modified alleles that include an internal ribosome entry site (IRES)-selectable marker cassette were expressed at lower levels than the wild-type GAPDH allele. ...
... GAPDH functions as a tetramer, and examination of the 3D crystal structure indicated that the C terminus of each GAPDH subunit is located on the exterior surface of the tetramer (Ismail and Park, 2005), suggesting that additional amino acids encoded by the T2A sequence should not interfere with enzymatic activity. However, examination of GAPDH protein using western blot analysis indicated that modified alleles that include an internal ribosome entry site (IRES)-selectable marker cassette were expressed at lower levels than the wild-type GAPDH allele. ...
... The second protein in the glycolytic pathway is glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This enzyme catalyzes the sixth step of glycolysis, during which a molecule of GA3-P is converted to 1,3-bisphosphoglycerate with production of NADH (130). Although GAPDH also possesses other functions, including DNA/RNA binding, gene transcription, and protein interaction (34), its role in the glycolytic process is unquestionable. ...
Significance:
Alzheimer disease (AD), a multifactorial neurodegenerative disorder that represents the most disabling condition in the aged, shares many features of insulin resistance diseases, suggesting AD can be considered a metabolic disease, characterized by reduced insulin signaling, increased oxidative stress, pro-inflammatory cytokine activation, mitochondrial dysfunction, impaired energy metabolism, and altered protein homeostasis.
Recent advances:
Reduced glucose utilization and energy metabolism in AD has been associated with accumulation of Aβ and hyperphosphorylated tau, increased oxidative stress, and accumulation of unfolded/misfolded proteins. mTOR, which is aberrantly activated in AD from early stages, plays key roles during AD neurodegeneration by inhibiting insulin signaling and regulating protein homeostasis.
Critical issues:
It is likely that the concomitant and mutual alterations of energy metabolism - mTOR signaling - protein homeostasis might represent a self-sustaining triangle of harmful events triggering the degeneration and death of neurons and the development and progression of AD. Altered crosstalk among components of this "triangle of death", beyond altering redox homeostasis of the neuron, is exacerbated by increased levels of oxidative stress that impair key components of the pathways involved. Redox proteomics studies in human samples and animal models of AD led to the identification of oxidatively modified components of the pathways composing the "triangle of death", therefore, revealing the crucial role of oxidative stress in fueling this aberrant vicious cycle.
Future directions:
Restoration of the functions of the pathways targeted by oxidative damage might represent a promising therapeutic approach to slow onset or delay progression of AD in the aged population.
... GAPDH functions as a tetramer, and examination of the 3D crystal structure indicated that the C terminus of each GAPDH subunit is located on the exterior surface of the tetramer (Ismail and Park, 2005), suggesting that additional amino acids encoded by the T2A sequence should not interfere with enzymatic activity. However, examination of GAPDH protein using western blot analysis indicated that modified alleles that include an internal ribosome entry site (IRES)-selectable marker cassette were expressed at lower levels than the wild-type GAPDH allele. ...
The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.
... The structures of GAPDH from archaea, bacteria, and eukaryotes have been determined by x-ray crystallography, and show that the NAD+ cofactor is bound, despite not being added during protein purification. In mammalian GAPDH structures, two, 110 three, 77,107 or even four NAD + molecules have been observed [111][112][113] per homotetramer. In addition, we recently showed that GAPDH was present as a mixture of NAD + -bound and NAD + -free species in solution by nano-ESI/MS/MS studies. ...
The glycolytic protein, glyceraldehyde‐3‐phosphate dehydrogenase ( GAPDH ), has a vast array of extraglycolytic cellular functions, including interactions with nucleic acids. GAPDH has been implicated in the translocation of transfer RNA ( tRNA ), the regulation of cellular messenger RNA ( mRNA ) stability and translation, as well as the regulation of replication and gene expression of many single‐stranded RNA viruses. A growing body of evidence supports GAPDH–RNA interactions serving as part of a larger coordination between intermediary metabolism and RNA biogenesis. Despite the established role of GAPDH in nucleic acid regulation, it is still unclear how and where GAPDH binds to its RNA targets, highlighted by the absence of any conserved RNA ‐binding sequences. This review will summarize our current understanding of GAPDH ‐mediated regulation of RNA function. WIREs RNA 2016, 7:53–70. doi: 10.1002/wrna.1315
This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition
RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
... Les acides aminés importants pour la fonction glycolytique sont la Cys 152 et l'His 179 [127] : l'His 179 interagit via des ponts hydrogènes avec la Cys 152 [161]. La Val 240 est caractéristique de la GAPDH, puisqu'elle forme un pont hydrogène avec l'Asn 316 lui permettant d'interagir avec le noyau nicotinamine du NAD + [160]. ...
S23906-1 is a diacetate benzo-[b] acronycine derivative. Selected for its cytotoxic potential in vitro and its antitumoral activity in vivo, this very promising compound entered clinical trial in 2006-2007. This alkylating agent presents the peculiarity to interact with the nitrogen in position 2 (N2) of guanines, localized in the minor groove of the DNA, and to subsequently lead local opening of the double helix. The conformation of this compound is important for its activity. Indeed, the presence of the active acetate group in the S-orientation leads to a stronger destabilization of the DNA with regard to the R-orientation, in correlation with a stronger cytotoxicity and a better antitumoral activity of the S- rather than R-stereoisomer. However, the molecular mechanism leading to the death of treated tumoral cell lines remains poorly understood. To address the link between the destabilization of the DNA and the cytotoxic activity of the S23906-1, we tried to determine the nature of nuclear proteins that specifically recognize this lesion. A proteomic approach, using chromatographic affinity followed by SDS-PAGE electrophoresis and MALDI-TOF analysis, identified the GAPDH protein. EMSA experiments validated the interaction of GAPDH to the S23906-1/DNA adduct (as a single- or double-stranded DNA template). The search for a possible consensus DNA-binding sequence of GAPDH was performed using a CASTing method. Even though no consensus site was clearly identified, prevalence for G-rich and GT-rich sequences was evidenced. From structure/activity relationships studies, we showed that DNA alkylation using the diacetylated or benzo-[a]-diacetylated forms of acronycine also destabilize the DNA helix and that GAPDH also binds in an efficient manner to these adducts. By comparison to other compounds also interacting with the N2 group of guanines, no interaction of the GAPDH with the ET-743/ADN adduct was evidenced (ET-743 stabilizes the DNA), while other authors evidenced an interaction with DNA of QAD, a Saframycine A derivative and a molecule structurally related to ET-743. At the cellular level, cellular treatment with the QAD resulted in GAPDH translocation from the cytoplasm to the nucleus. We did not observe such a translocation using S23906-1. Furthermore cells treated with a siRNA directed to the GAPDH are more resistant to S23906-1, suggesting an anti-apoptotic role of GAPDH after S23906-1 treatment, by contrast with the pro-apoptotic effect observed in the literature with QAD . In parallel, we were interested in new cytotoxic agents, among which abis-8-hydroxyquinoline benzylamine series. Using spectrometric and biochemical approaches, we showed that the lead compound JLK1486 does not target DNA, but covalently reacts with thiol groups as that of the glutathione. However, cellular studies using depletion of the endogenous glutathione showed that the glutathione was not the target of JLK1486 but that it participated in its detoxification as it does for S23906-1. To identify the protein(s) target(s) responsible for the cytotoxic effect of JLK1486, we developed an approach derived from the 2D-DIGE electrophoresis which clearly evidenced proteins that are the cellular targets for JLK1486.
... Każda podjednostka GAPDH złożona jest z pojedynczego łańcucha polipeptydowego zbudowanego z 335 reszt aminokwasowych. Badania strukturalne pozwoliły wyodrębnić w podjednostkach dwie domeny funkcjonalne: domenę katalityczną (reszty aminokwasowe 152-314) oraz domenę wiążącą koenzym NAD + (reszty aminokwasowe 1-151, 315-335) [31,32,46] (ryc. 1). ...
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key redox-sensitive protein, the activity of which is largely affected by oxidative modifications at its highly reactive cysteine residue in the active site of the enzyme (Cys-152). These modifications occur as a result of S-thiolation, S-nitrosylation or disulfide bonds that lead to aggregate formation. The oxidative changes not only affect the glycolytic function but also stimulate the participation of GAPDH in numerous cellular processes. In this review we describe how thiol modification of Cys-152 in GAPDH re-routes metabolic pathways in the cell and converts a metabolic enzyme into a pro-apoptotic factor. Especially interesting issue is the participation of GAPDH in the regulation of expression of endothelin 1 and nitrosylation of nuclear proteins. In the last section we describe involvement of GAPDH in the processes associated with neurodegenerative diseases.
... In this pathway, GAPDH stands out as one of the main targets, as it is a key enzyme in this process [12]. Humans also possess GAPDH that could be at least partially inhibited by nonselective compounds [13]. However, it is claimed that no toxic effect in human cells would be observed even when more than 95% of human GAPDH (hGAPDH) is inhibited [14]. ...
Background:
The enzyme gapdh, which acts in the glycolytic pathway, is seen as a potential target for pharmaceutical intervention of chagas disease.
Results:
Herein, we report the discovery of new Trypanosoma cruzi GAPDH (TcGAPDH) inhibitors from target- and ligand-based virtual screening protocols using isothermal titration calorimetry (ITC) and molecular dynamics. Molecular dynamics simulations were used to gain insight on the binding poses of newly identified inhibitors acting at the TcGAPDH substrate (G3P) site.
Conclusion:
Nequimed125, the most potent inhibitor to act upon TcGAPDH so far, which sits on the G3P site without any contact with the co-factor (NAD(+)) site, underpins the result obtained by ITC that it is a G3P-competitive inhibitor. Molecular dynamics simulation provides biding poses of TcGAPDH inhibitors that correlate with mechanisms of inhibition observed by ITC. Overall, a new class of dihydroindole compounds that act upon TcGAPDH through a competitive mechanism of inhibition as proven by ITC measurements also kills T. cruzi.
... The initial step of this reaction is dependent upon Cys152 within the GAPDH active site (Fig. 7), which mediates nucleophilic attack on the G3P carbonyl to yield a hemithioacetal intermediate. The IISNASCTTNCLAPLAK peptide identified in our study is located within the C-terminal catalytic domain of GAPDH 44 and contained ACR adducts on Cys152 and Cys156. Because Cys152 plays a critical role in GAPDH enzyme function, ACR adduction of this residue likely mediates dysfunction. ...
α,β-Unsaturated carbonyls make up an important class of chemicals involved in environmental toxicity and disease processes. Whereas adduction of cysteine residues on proteins is a well-documented reaction of these chemicals, such a generic effect cannot explain the molecular mechanism of cytotoxicity. Instead, more detailed information is needed regarding the possible specificity and kinetics of cysteine targeting and the quantitative relationship between adduct burden and protein dysfunction. To address these data gaps, we incubated purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with acrylamide (ACR), acrolein, or methylvinyl ketone (MVK). Results show that these α,β-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration- and time-dependent manner. The rank order of enzyme inhibition (K(I)) (i.e., ACR ≪ MVK < acrolein) was related to the calculated electrophilic reactivity of each compound and to the corresponding kinetics of cysteine adduct formation. Tandem mass spectrometry revealed that adduct formation was selective at lower concentrations; i.e., ACR preferentially formed adducts with Cys152 (residues 146-162). At higher concentrations, ACR also formed adducts with Cys156 and Cys247 (residues 235-248). Adduct formation at Cys152 was correlated to enzyme inhibition, which is consistent with the regulatory role of this residue in enzyme function and its location within the GAPDH active site. Further analyses indicated that Cys152 was present in a pK(a)-lowering microenvironment (pK(a) = 6.03), and at physiological pH, the corresponding sulfhydryl group exists in the highly reactive nucleophilic thiolate state. These data suggest a general cytotoxic mechanism in which electrophilic α,β-unsaturated carbonyls selectively form adducts with reactive nucleophilic cysteine residues specifically associated with the active sites of proteins. These specialized cysteine residues are toxicologically relevant molecular targets, because chemical derivatization causes loss of protein function.
... GAPDH, via a redox reaction, converts glyceraldehyde-3phosphate into 1,3-biphosphoglycerate with concomitant reduction of nicotinamide adenine dinucleotide (NAD1) to NADH. The enzyme is a homotetramer, and each subunit of it may bind to NAD1 [37,70]. As with other enzymes, GAPDH is a multifunctional protein with glycolytic and non-glycolytic functions, both of which can be of oncological interest. ...
Most cancer cells exhibit elevated levels of glycolysis and this metabolic pathway seems to be related to a greater glucose uptake. This phenomenon, known as the Warburg effect, is considered one of the most fundamental metabolic alterations during malignant transformation. Originally, Warburg hypothesised that the aerobic glycolysis of cancer cells could be just an aspect of a more complex metabolic adaptation. However, this intriguing discovery was partially misinterpreted and disregarded over time. In recent years, the peculiarities of cancer cell metabolism have been re-evaluated in light of new metabolic data that seem to confirm and to widen the original concept of the Warburg effect. In fact, biochemical, molecular, and, above all, proteomic studies on the multifaceted roles of glycolytic enzymes in cancer cells in general, and in cancer stem cells in particular, seem to suggest more complex functional adaptations. These adaptations result in significantly altered protein expression patterns, and they have fundamental implications for diagnosis, prognosis and therapy. Revisiting the Warburg effect in cancer cells with a proteomic approach could deepen our knowledge of cancer cell metabolism and of cancer cell biology in general. Moreover, by identifying useful diagnostic, prognostic and therapeutic targets, it could significantly impact clinical practice.
... Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), by means of a redox reaction, converts glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate with concomitant reduction of nicotinamide adenine dinucleotide (NAD + ) to NADH. The enzyme structure consists of a homotetramer, each subunit of which may bind to NAD + [31,76] . Like other enzymes, GAPDH is a multifunctional protein with glycolytic and nonglycolytic functions, both of which can be of chemotherapeutic value. ...
The radio- and chemotherapeutics currently used for the treatment of cancer are widely known to be characterized by a low therapeutic index. An interesting approach to overcoming some of the limits of these techniques is the exploitation of the so-called Warburg effect, which typically characterizes neoplastic cells. Interestingly, this feature has already been utilized with good results, but only for diagnostic purposes (PET and SPECT). From a pharmacological point of view, drugs able to perturb cancer cell metabolism, specifically at the level of glycolysis, may display interesting therapeutic activities in cancer.
The pharmacological actions of these glycolytic enzyme inhibitors, based primarily on ATP depletion, could include: i) amelioration of drug selectivity by exploiting the particular glycolysis addiction of cancer cell; ii) inhibition of energetic and anabolic processes; iii) reduction of hypoxia-linked cancer-cell resistance; iv) reduction of ATP-dependent multi-drug resistance; and v) cytotoxic synergism with conventional cancer treatments.
Several glycolytic inhibitors are currently in preclinical and clinical development. Their clinical value as anticancer agents, above all in terms of therapeutic index, strictly depends on a careful reevaluation of the pathophyiological role of the unique metabolism of cancer cells in general and of Warburg effect in particular.
The process of heme binding to a protein is prevalent in almost all forms of life to control many important biological properties, such as O2-binding, electron transfer, gas sensing or to build catalytic power. In these cases, heme typically binds tightly (irreversibly) to a protein in a discrete heme binding pocket, with one or two heme ligands provided most commonly to the heme iron by His, Cys or Tyr residues. Heme binding can also be used as a regulatory mechanism, for example in transcriptional regulation or ion channel control. When used as a regulator, heme binds more weakly, with different heme ligations and without the need for a discrete heme pocket. This makes the characterization of heme regulatory proteins difficult, and new approaches are needed to predict and understand the heme-protein interactions. We apply a modified version of the ProFunc bioinformatics tool to identify heme-binding sites in a test set of heme-dependent regulatory proteins taken from the Protein Data Bank and AlphaFold models. The potential heme binding sites identified can be easily visualized in PyMol and, if necessary, optimized with RosettaDOCK. We demonstrate that the methodology can be used to identify heme-binding sites in proteins, including in cases where there is no crystal structure available, but the methodology is more accurate when the quality of the structural information is high. The ProFunc tool, with the modification used in this work, is publicly available at https://www.ebi.ac.uk/thornton-srv/databases/profunc and can be readily adopted for the examination of new heme binding targets.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme, plays a crucial role in the energy metabolism of cancer cells and has been proposed as a valuable target for the development of anticancer agents. Among a series of 5-substituted 3-bromo-4,5-dihydroisoxazole (BDHI) derivatives, we identified the spirocyclic compound 11, which is able to covalently inactivate recombinant human GAPDH (hGAPDH) with a faster reactivity than koningic acid, one of the most potent hGAPDH inhibitors known to date. Computational studies confirmed that conformational rigidification is crucial to stabilize the interaction of the inhibitor with the binding site, thus favoring the subsequent covalent bond formation. Investigation of intrinsic warhead reactivity at different pH disclosed the negligible reactivity of 11 with free thiols, highlighting its ability to selectively react with the activated cysteine of hGAPDH with respect to other sulfhydryl groups. Compound 11 strongly reduced cancer cell growth in four different pancreatic cancer cell lines and its antiproliferative activity correlated well with the intracellular inhibition of hGAPDH. Overall, our results qualify 11 as a potent hGAPDH covalent inhibitor with a moderate drug-like reactivity that could be further exploited to develop anticancer agents.
Program and list of Abstracts for the communications presented in the 7th International Iberian Biophysics Congress, orally and as poster.
The meeting took place between 14 and 16 June 2021, in an online format organized at the University of Coimbra, Coimbra, Portugal.
Background : n-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyses the NAD ⁺ -dependent oxidative phosphorylation of n-glyceraldehyde-3-phosphate to 1,3-diphospho-n-glycerate and its reverse reaction in glycolysis and gluconeogenesis.
Methods : Four distinct crystal structures of human n-Glyceraldehyde-3-phosphate dehydrogenase ( Hs GAPDH) have been determined from protein purified from the supernatant of HEK293F human epithelial kidney cells.
Results : X-ray crystallography and mass-spectrometry indicate that the catalytic cysteine of the protein ( Hs GAPDH Cys152) is partially oxidised to cysteine S-sulfonic acid. The average occupancy for the Cys152-S-sulfonic acid modification over the 20 crystallographically independent copies of Hs GAPDH across three of the crystal forms obtained is 0.31±0.17.
Conclusions : The modification induces no significant structural changes on the tetrameric enzyme, and only makes aspecific contacts to surface residues in the active site, in keeping with the hypothesis that the oxidising conditions of the secreted mammalian cell expression system result in Hs GAPDH catalytic cysteine S-sulfonic acid modification and irreversible inactivation of the enzyme.
Overcoming resistance to bioactive small molecules is a significant challenge for health care and agriculture. Therefore, efforts to uncover the mechanisms of resistance are essential to the development of new antibiotics, anticancer drugs and pesticides. To study how nature evolves resistance to highly potent natural products, we examined the biosynthesis and mechanism of self-resistance of the fungal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor heptelidic acid (HA). HA is a nanomolar inhibitor of GADPH through the covalent modification of the active site cysteine thiol. The biosynthetic pathway of HA was elucidated, which uncovered the enzymatic basis of formation of the epoxide warhead. Structure-activity relationship study using biosynthetic intermediates established the importance of the fused lactone ring system in HA. The molecular mode of action of HA inhibiting human GAPDH was illustrated through the crystal structure of Hs-GAPDH covalently bound with HA. A GAPDH isozyme HepG encoded in HA cluster was characterized to be less sensitive to HA, and therefore confer self-resistance for the producing host. Comparison of the crystal structures of human GAPDH and HepG showed mutations both within and remote to the active site can contribute to resistance of inactivation, which were confirmed through mutagenesis. Due to the critical role GAPDH plays in aerobic glycolysis and others, knowledge of the mode of action of HA and self-resistance mechanism could accelerate the development of improved inhibitors.
Background : n-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyses the reversible NAD ⁺ -dependent oxidative phosphorylation of n-glyceraldehyde-3-phosphate to 1,3-diphospho-n-glycerate in both glycolysis and gluconeogenesis.
Methods : Four distinct crystal structures of human n-Glyceraldehyde-3-phosphate dehydrogenase ( Hs GAPDH) have been determined from protein purified from the supernatant of HEK293F human epithelial kidney cells.
Results : X-ray crystallography and mass-spectrometry indicate that the catalytic cysteine of the protein ( Hs GAPDH Cys152) is partially oxidised to cysteine S-sulfonic acid. The average occupancy for the Cys152-S-sulfonic acid modification over the 20 crystallographically independent copies of Hs GAPDH across three of the crystal forms obtained is 0.31±0.17.
Conclusions : The modification induces no significant structural changes on the tetrameric enzyme, and only makes aspecific contacts to surface residues in the active site, in keeping with the hypothesis that the oxidising conditions of the secreted mammalian cell expression system result in Hs GAPDH catalytic cysteine S-sulfonic acid modification and irreversible inactivation of the enzyme.
Naegleria gruberi is a free life amoeba believed to have more than one billion years of existence; it is not pathogenic and had its genome sequenced, which revealed a high complexity in the metabolic pathways. This paper presents the experimental structure of GAPDH from N. gruberi, the first one belonging to the phylum Percolozoa, comparisons to structures from various species and molecular dynamics studies of some particular features. The final refined structure presents Rcryst = 15.54% and Rfree = 19.84%. The catalytic domain formed by residues 134 to 313 is highly conserved, as expected, with the exception of Asn145, present only in NgGAPDH, while the other GAPDHs present either Ser or Thr on the corresponding position. Molecular dynamics analysis revealed that Asn145 has correlated motions with residues Ala123, Thr125 and Pro126 that belong to what was called "bonded loop". NgGAPDH residue Met35 presents an extended side chain, closer to the cofactor adenine ring than corresponding (different) residues and conformations found in some parasitic protozoa and the human GAPDHs. The enzyme was previously reported to present positive cooperativity, which is hypothesized to be related to certain atom distances.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (BGP) in the presence of the NAD cofactor. GAPDH is an important drug target because of its central role in glycolysis, and non-glycolytic processes such as nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis. Recent studies found that GAPDH participates in the development of diabetic retinopathy and its progression after the cessation of hyperglycemia. Here, we report two structures for native bovine photoreceptor GAPDH as a homotetramer with differing occupancy by NAD, bGAPDH(NAD)4 and bGAPDH(NAD)3. The bGAPDH(NAD)4 was solved at 1.52 Å, the highest resolution for GAPDH. Structural comparison of the bGAPDH(NAD)4 and bGAPDH(NAD)3 models revealed novel details of conformational changes induced by cofactor binding, including a loop region (residues 54 – 56). Structure analysis of bGAPDH confirmed the importance of Phe34 in NAD binding, and demonstrated that Phe34 was stabilized in the presence of NAD but displayed greater mobility in its absence. The oxidative state of the active site Cys149 residue is regulated by NAD binding, because this residue was found oxidized in the absence of dinucleotide. The distance between Cys149 and His176 decreased upon NAD binding and Cys149 remained in a reduced state when NAD was bound. These findings provide an important structural step for understanding the mechanism of GAPDH activity in vision and its pathological role in retinopathies.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis and shown, particularly in animal cells, to play additional roles in several unrelated non-metabolic processes such as control of gene expression and apoptosis. This functional versatility is regulated, in part at least, by redox post-translational modifications that alter GAPDH catalytic activity and influence the subcellular localization of the enzyme. In spite of the well established moonlighting (multifunctional) properties of animal GAPDH, little is known about non-metabolic roles of GAPDH in plants. Plant cells contain several GAPDH isoforms with different catalytic and regulatory properties, located both in the cytoplasm and in plastids, and participating in glycolysis and the Calvin-Benson cycle. A general feature of all GAPDH proteins is the presence of an acidic catalytic cysteine in the active site that is overly sensitive to oxidative modifications, including glutathionylation and S-nitrosylation. In Arabidopsis, oxidatively modified cytoplasmic GAPDH has been successfully used as a tool to investigate the role of reduced glutathione, thioredoxins and glutaredoxins in the control of different types of redox post-translational modifications. Oxidative modifications inhibit GAPDH activity, but might enable additional functions in plant cells. Mounting evidence support the concept that plant cytoplasmic GAPDH may fulfill alternative, non-metabolic functions that are triggered by redox post-translational modifications of the protein under stress conditions. The aim of this review is to detail the molecular mechanisms underlying the redox regulation of plant cytoplasmic GAPDH in the light of its crystal structure, and to provide a brief inventory of the well known redox-dependent multi-facetted properties of animal GAPDH, together with the emerging roles of oxidatively modified GAPDH in stress signaling pathways in plants.
The development of fast and reliable methods for the identification of new bioactive compounds is of utmost importance to boost the process of drug discovery and development. Immobilized enzyme reactors (IMERs), integrated with high performance liquid chromatography (HPLC), are attractive and versatile tools for screening collections consisting of natural products and synthetic small molecules. Standard kinetic parameters of the immobilized enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from both Trypanosoma cruzi de and human have been determined (T. cruzi: K-M(G3P) = 0.50 mmol L-1; K-M(NAD+) = 0.67 mmol L-1; humana: K-M(G3P) = 3.7 mmol L-1; K-M(NAD+) = 0.75 mmol L-1), and comparisons of these values with those of the parasite and human free enzymes indicate a decrease in the affinity for the immobilized system (T. cruzi: K-M(G3P) = 0.42 mmol L-1; K-M(NAD+) = 0.26 mmol L-1; humana: K-M(G3P) = 0.16 mmol L-1; K-M(NAD+) = 0.18 mmol L-1). Interestingly, despite the kinetic differences between the two systems, the immobilized GAPDHs retained the required structural requirements for molecular recognition and biological activity, increasing the stability the enzyme. In the present work, we described an integrated structural analysis which has provided important insights into the molecular basis underlying the effects of immobilization on the ligand-receptor interactions and consequent enzymatic activity and kinetics parameters.
hGAPDS (human sperm-specific glyceraldehyde-3-phosphate dehydrogenase) is a glycolytic enzyme essential for the survival of spermatozoa, and constitutes a potential target for non-hormonal contraception. However, enzyme characterization of GAPDS has been hampered by the difficulty in producing soluble recombinant protein. In the present study, we have overexpressed in Escherichia coli a highly soluble form of hGAPDS truncated at the N-terminus (hGAPDSΔN), and crystallized the homotetrameric enzyme in two ligand complexes. The hGAPDSΔN-NAD+-phosphate structure maps the two anion-recognition sites within the catalytic pocket that correspond to the conserved Ps site and the newly recognized Pi site identified in other organisms. The hGAPDSΔN-NAD+-glycerol structure shows serendipitous binding of glycerol at the Ps and new Pi sites, demonstrating the propensity of these anion-recognition sites to bind non-physiologically relevant ligands. A comparison of kinetic profiles between hGAPDSΔN and its somatic equivalent reveals a 3-fold increase in catalytic efficiency for hGAPDSΔN. This may be attributable to subtle amino acid substitutions peripheral to the active centre that influence the charge properties and protonation states of catalytic residues. Our data therefore elucidate structural and kinetic features of hGAPDS that might provide insightful information towards inhibitor development.
Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein. A structure was also obtained where glyceraldehyde 3-phosphate binds in the P(s) pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of alpha-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives.
Multiple lines of evidence shows that tumorigenesis is often associated with altered carbohydrate metabolism, characterized by increased glucose uptake and elevated glycolysis, which was first recognized by Otto Warburg 70 years ago. Therefore, the inhibition of glycolysis has been proposed as a therapeutic strategy for cancer treatment. However, this disordered glycotic process does not represent the whole picture of altered energy metabolism during cancer cell transformation. In order to achieve rapid cell proliferation, tumor cells have to constantly accumulate large amount of macromolecules for replication, which has led to several hallmarks of cancer demonstrating its robust metabolic adaptation, including high levels of aerobic glycolysis rate, high rate of energy-consuming processes for syntheses of proteins, DNA and fatty acids. This review summarizes some potential drugable targets as well as their pharmacological inhibitors in glucose, glutamine and fatty acid metabolic pathways. In addition, the upstream oncogenic signaling pathways that are tightly in conjunction with the altered metabolism in tumors are also covered.
The presence of an isoform of glyceraldehyde-3-phosphate dehydrogenase (kmGAPDH1p) associated with the cell wall of a flocculent
strain of Kluyveromyces marxianus was the first report of a non-cytosolic localization of a glycolytic enzyme, but the mechanism by which the protein is transported
to the cell surface is not known. To identify structural features that could account for the multiple localizations of the
protein, the three-dimensional structure of kmGAPDH1p was determined by x-ray crystallography and small angle x-ray scattering.
The x-ray crystallographic structure of kmGAPDH1p revealed a dimer, although all GAPDH homologs studied thus far have a tetrameric
structure with 222 symmetry. Interestingly, the structure of kmGAPDH1p in solution revealed a tetramer with a 70° tilt angle
between the dimers. Moreover, the separation between the centers of the dimers composing the kmGAPDH1p tetramer diminished
from 34 to 30 Å upon NAD+ binding, this latter value being similar to the observed in the crystallographic models of GAPDH homologs. The less compact
structure of apo-kmGAPDH1p could already be the first image of the transition intermediate between the tetramer observed in
solution and the dimeric form found in the crystal structure, which we postulate to exist in vivo because of the protein's multiple subcellular localizations in this yeast species.
This study was undertaken to identify cellular proteins that bind an orally active natural product insulin mimic. Phage display cloning was used with a biotinylated derivative of this molecule as bait. Among the proteins identified was glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which has recently been shown to affect insulin receptor signaling. Binding data support a role for human GAPDH as another target of the insulin mimic, which could explain its action as a selective insulin receptor modulator.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the pathogenic protozoa Entamoeba histolytica (Eh) is a major glycolytic enzyme and an attractive drug target since this parasite lacks a functional citric acid cycle and is dependent solely on glycolysis for its energy requirements. The three-dimensional structure of dimeric EhGAPDH in complex with cofactor NAD(+) has been generated by homology modeling based on the crystal structure of human liver GAPDH. Our refined model indicates the presence of a parasite specific disulfide bond between two cysteine residues of adjacent monomers in the EhGAPDH dimer, which may be an important target for future drug design. Flexible docking with the substrate glyceraldehyde-3-phosphate (G3P) shows that Cys151, His178, Thr210, and Arg233 are important residues in G3P binding. The inorganic phosphate-binding site of EhGAPDH has been determined by docking study. The binding mode of a natural GAPDH inhibitor, chalepin to EhGAPDH has also been predicted. In search for a better inhibitor for EhGADPH, in silico modification of chalepin has been carried out to form an additional specific polar interaction with Asp194 of EhGAPDH whose equivalent is Leu195 in human GAPDH. In the absence of a crystal structure, our study provides an early insight into the structure of major drug target EhGAPDH, thus, facilitating the inhibitor design.
The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is as an attractive target for the development of novel antitrypanosomatid agents. In the present work, comparative molecular field analysis and comparative molecular similarity index analysis were conducted on a large series of selective inhibitors of trypanosomatid GAPDH. Four statistically significant models were obtained ( r2 > 0.90 and q2 > 0.70), indicating their predictive ability for untested compounds. The models were then used to predict the potency of an external test set, and the predicted values were in good agreement with the experimental results. Molecular modeling studies provided further insight into the structural basis for selective inhibition of trypanosomatid GAPDH.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of pathogenic protozoa Trichomonas vaginalis (TvGAPDH) is an attractive drug target since this parasite lacks functional citric acid cycle and is dependent solely on glycolysis for its energy requirements. The three dimensional structure of TvGAPDH dimer has been generated by homology modelling based on the crystal structure of human liver GAPDH. Comparison of the NAD;{+} binding pocket of the modeled TvGAPDH with human GAPDH (hGAPDH) reveals the presence of a hydrophobic pocket near the N-6 position of adenine ring as well as a hydrophobic cleft near O-2' of the adenosine ribose that are absent in the human enzyme. In order to exploit these structural differences adenosine and several adenosine analogs with substitution on N-6 position of adenine ring or 2' position of ribose sugar or both have been studied by docking experiments using the program AutoDock version 3.0.5. Our docking result suggests that bulkier hydrophobic substitution at the N-6 position of the adenine ring could form more stable complexes with TvGAPDH than with hGAPDH. An improvement of binding occurs in TvGAPDH when methoxybenzamido group has been introduced at the O-2' position of the ribose sugar. The combination of N-6 and O-2' substitutions may have produced significantly improved inhibitors. Our study may help in identifying structural elements involved in the origin of selectivity at the NAD;{+} binding pocket of TvGAPDH. This study could further be extended for future anti-trichomonal drug design strategies in order to control trichomoniasis.
Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ 2 ) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
Within the framework of a project aimed at rational design of drugs against diseases caused by trypanosomes and related hemoflagellate parasites, selective inhibitors of trypanosomal glycolysis were designed, synthesized, and tested. The design was based upon the crystallographically determined structures of the NAD:glyceraldehyde-3-phosphate dehydrogenase complexes of humans and Trypanosoma brucei, the causative agent of sleeping sickness. After one design cycle, using the adenosine part of the NAD cofactor as a lead, the following encouraging results were obtained: (1) a 2-methyl substitution, targeted at a small pocket near Val 36, improves inhibition of the parasite enzyme 12.5-fold; (2) an 8-(thien-2-yl) substitution, aimed at Leu 112 of the parasite enzyme, where the equivalent residue in the mammalian enzyme is Val 100, results in a 167-fold better inhibition of the trypanosomal enzyme, while the inhibition of the human enzyme is improved only 13-fold; (3) exploitation of a "selectivity cleft" created by a unique backbone conformation in the trypanosomal enzyme near the adenosine ribose yields a considerable improvement in selectivity: 2'-deoxy-2'-(3-methoxybenzamido)adenosine inhibits the human enzyme only marginally but enhances inhibition of the parasite enzyme 45-fold when compared with adenosine. The designed inhibitors are not only better inhibitors of T. brucei GAPDH but also of the enzyme from Leishmania mexicana.
The three-dimensional structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.12.1.12] from the sleeping-sickness parasite Trypanosoma brucei was solved by molecular replacement at 3.2-A resolution with an x-ray data set collected by the Laue method. For data collection, three crystals were exposed to the polychromatic synchrotron x-ray beam for a total of 20.5 sec. The structure was solved by using the Bacillus stearothermophilus enzyme model [Skarzyński, T., Moody, P. C. E. & Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187] with a partial data set which was 37% complete. The crystals contain six subunits per asymmetric unit, which allowed us to overcome the absence of > 60% of the reflections by 6-fold density averaging. After molecular dynamics refinement, the current molecular model has an R factor of 17.6%. Comparing the structure of the trypanosome enzyme with that of the homologous human muscle enzyme, which was determined at 2.4-A resolution, reveals important structural differences in the NAD binding region. These are of great interest for the design of specific inhibitors of the parasite enzyme.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered a classical glycolytic protein examined for its pivotal role in energy production. It was also used as a model protein for analysis of protein structure and enzyme mechanisms. The GAPDH gene was utilized as a prototype for studies of genetic organization, expression and regulation. However, recent evidence demonstrates that mammalian GAPDH displays a number of diverse activities unrelated to its glycolytic function. These include its role in membrane fusion, microtubule bundling, phosphotransferase activity, nuclear RNA export, DNA replication and DNA repair. These new activities may be related to the subcellular localization and oligomeric structure of GAPDH in vivo. Furthermore, other investigations suggest that GAPDH is involved in apoptosis, age-related neurodegenerative disease, prostate cancer and viral pathogenesis. Intriguingly, GAPDH is also a unique target of nitric oxide. This review discusses the functional diversity of GAPDH in relation to its protein structure. The mechanisms through which mammalian cells may utilize GAPDH amino acid sequences to provide these new functions and to determine its intracellular localization are considered. The interrelationship between new GAPDH activities and its role in cell pathologies is addressed.
The rate of glucose transport across the plasma membrane of the bloodstream form of Trypanosoma brucei was modulated by titration of the hexose transporter with the inhibitor phloretin, and the effect on the glycolytic flux was measured. A rapid glucose uptake assay was developed to measure the transport activity independently of the glycolytic flux. Phloretin proved a competitive inhibitor. When the effect of the intracellular glucose concentration on the inhibition was taken into account, the flux control coefficient of the glucose transporter was between 0.3 and 0.5 at 5 mM glucose. Because the flux control coefficients of all steps in a metabolic pathway sum to 1, this result proves that glucose transport is not the rate-limiting step of trypanosome glycolysis. Under physiological conditions, transport shares the control with other steps. At glucose concentrations much lower than physiological, the glucose carrier assumed all control, in close agreement with model predictions.
The crystal structure of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus was solved in complex with its cofactor, NAD, and its physiological substrate,d-glyceraldehyde 3-phosphate (d-G3P). To isolate a stable ternary complex, the nucleophilic residue of the active site, Cys149, was substituted with alanine or serine. The C149A and C149S GAPDH ternary complexes were obtained by soaking the crystals
of the corresponding binary complexes (enzyme·NAD) in a solution containing G3P. The structures of the two binary and the
two ternary complexes are presented. The d-G3P adopts the same conformation in the two ternary complexes. It is bound in a non-covalent way, in the free aldehyde form,
its C-3 phosphate group being positioned in the Ps site and not in the Pi site. Its C-1 carbonyl oxygen points toward the essential His176, which supports the role proposed for this residue along the two steps of the catalytic pathway. Arguments are provided that
the structures reported here are representative of a productive enzyme·NAD·d-G3P complex in the ground state (Michaelis complex).
The crystal structure of the tetrameric form of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isolated from rabbit muscle was solved at 2.4 A resolution after careful dynamic light-scattering experiments to find a suitable buffer for crystallization trials. The refined model has a crystallographic R factor of 20.3%. Here, the first detailed model of a mammalian GAPDH is presented. The cofactor NAD(+) (nicotinamide adenine dinucleotide) is bound to two subunits of the tetrameric enzyme, which is consistent with the negative cooperativity of NAD(+) binding to this enzyme. The structure of rabbit-muscle GAPDH is of interest because it shares 91% sequence identity with the human enzyme; human GAPDH is a potential target for the development of anti-apoptotic drugs. In addition, differences in the cofactor-binding pocket compared with the homology-model structure of GAPDH from the malaria parasite Plasmodium falciparum could be exploited in order to develop novel selective and potential antimalaria drugs.
The three-dimensional crystal structure of the enzyme glyceraldehyde phosphate dehydrogenase from the kinetoplastid Trypanosoma brucei brucei has been determined at 3.2 A resolution from a 37% complete data set collected using the Laue method. The crystals used in the structure determination contain one and a half tetrameric enzyme molecules in the asymmetric unit, i.e. six identical subunits. Initial phasing was carried out by the method of molecular replacement using the refined coordinates of holo glyceraldehyde phosphate dehydrogenase from Bacillus stearothermophilus as a search model. The initial electron-density distribution, obtained from the molecular-replacement solution, was greatly improved by a procedure consisting of 36 cycles of iterative non-crystallographic density averaging. During the averaging procedure, the missing reflections (63% of the data) were gradually introduced as map-inversion structure factors. At completion of the procedure, the R-factor between averaged map-inversion amplitudes and observed structure-factor amplitudes was 19.0% for all data between 7.0 and 3.2 A resolution, and that between the map-inversion amplitudes and later recorded structure-factor amplitudes was 41.9%. After model building into the resulting averaged electron-density map, refinement by molecular-dynamics procedures with X-PLOR provided the current model, which has an R-factor of 17.6% for 34 835 reflections between 7.0 and 3.2 A resolution. The refined model, comprising 2735 protein atoms plus one NAD(+) molecule and two sulfate ions per subunit, has r.m.s. deviations from ideality of 0.02 A for bond lengths and 3.6 degrees for bond angles. All subunits, located either within the tetrameric molecule or within the half tetramer present in the asymmetric unit, are related to each other by almost exact twofold symmetry. The overall structure of the glycosomal glyceraldehyde phosphate dehydrogenase subunit and its quaternary arrangement in the tetrameric molecule are similar to that of the enzyme of lobster and Bacillus stearothermophilus (with r.m.s. differences between equivalent Calpha positions of 0.71 and 0.64 A, respectively). The main differences between the structures is the presence of three insertions, plus the substitution of a beta-strand by a short alpha-helix, both occurring at the surface of the glycosomal enzyme subunit.
Recent articles have highlighted numerous additional functions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that are independent of its well-documented glycolytic function. One of the most intriguing of these functions is as an initiator of programmed cell death cascades. This activity involves a nuclear appearance of GAPDH, a considerable proportion of which requires synthesis of new GAPDH protein and has characteristics suggesting the involvement of a novel isozyme. The relevance of such findings to human neurodegenerative conditions is emphasized by the increased nuclear GAPDH observed in postmortem samples from patients with Parkinson's disease, Alzheimer's disease, Huntington's disease and glaucoma, among others. A number of small-molecule compounds have now been identified that show anti-apoptotic activity because of their ability to interact with GAPDH and prevent its nuclear accumulation. These compounds, one of which is currently being tested in late-stage Phase II clinical trials as a disease-modifying therapy for Parkinson's disease, have potential utility in the treatment of human neurodegenerative conditions.
: Abundant senile plaques are a histological hallmark in the brain of Alzheimer's disease patients. Such plaques consist of, among many other constituents, aggregated βA4 amyloid peptide. This peptide is derived from an amyloid precursor protein (APP) by irregular proteolytic processing and is considered to be involved in the development of Alzheimer's disease. To study possible interactions of brain proteins with 0A4 amyloid or other fragments of APP, βA4 amyloid and βA4 amyloid extended to the C-terminus of APP were recombinantly produced as fusion proteins termed “Amy” and “AmyC,” respectively. Using Amy and AmyC affinity chromatography, a 35-kDa protein from rat brain was isolated that bound tightly to AmyC but not to Amy, thus indicating an interaction of the protein with the C-terminus of APP. This 35-kDa protein was identified as the glycolytic enzyme gIyceraldehyde-3-phosphate dehydrogenase (GAPDH). Binding of GAPDH to AmyC but not to Amy was confirmed by gel filtration. Although AmyC slightly reduced the Vmax of GAPDH, the same reduction was observed in the presence of Amy. These findings suggest that the interaction of the cytoplasmic domain of APP with GAPDH is unlikely to influence directly the rate of glycolysis but may serve another function.
In continuation of a project aimed at the structure-based design of drugs against sleeping sickness, analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine (1) were synthesized and tested to establish structure-activity relationships for inhibiting glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Compound 1 was recently designed using the NAD:GAPDH complexes of the human enzyme and that of Trypanosoma brucei, the causative agent of sleeping sickness. In an effort to exploit an extra hydrophobic domain due to Val 207 of the parasite enzyme, several new 2'-amido-2'-deoxyadenosines were synthesized. Some of them displayed an interesting improvement in inhibitory activity compared to 1. Carbocyclic or acyclic analogs showed marked loss of activity, illustrating the importance of the typical (C-2'-endo) puckering of the ribose moiety. We also describe the synthesis of a pair of compounds that combine the beneficial effects of a 2- and 8-substituted adenine moiety on potency with the beneficial effect of a 2'-amido moiety on selectivity. Unfortunately, in both cases, IC50 values demonstrate the incompatibility of these combined modifications. Finally, introduction of a hydrophobic 5'-amido group on 5'-deoxyadenosine enhances the inhibition of the protozoan enzyme significantly, although the gain in selectivity is mediocre.
Phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GraP-DH) catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate to form 1.3-diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. Two essential aminoacids, Cys149 and His176 are involved in the chemical mechanism of bacterial and eukaryotic GraP-DHs. Roles have been assigned to the His176 as (a) a chemical activator for enhancing the reactivity of Cys149, (b) a stabilizator of the tetrahedral transition states, and (c) a base catalyst facilitating hydride transfer towards NAD. In a previous study carried out on Escherichia coli GraP-DH [Soukri, A., Mougin, A., Corbier, C., Wonacott, A. J., Branlant, C. & Branlant, G. (1989) Biochemistry, 28, 2586-2592], the role of His176 as an activator of the reactivity of Cys149 was studied. Here, we further investigated the role of the His residue in the chemical mechanism of phosphorylating GraP-DH from E. coli and Bacillus stearothermophilus. The chemical reactivity of Cys149 in the His176Asn mutant was reinvestigated. At neutral pH, its reactivity was shown to be at least as high as that observed in the Cys-/His+ ion pair present in the wild type. No pre-steady state burst of NADH was found with the His176Asn mutant in contrast to what is observed for the wild type, and a primary isotope effect was observed when D-[1-2H]glyceraldehyde-3-phosphate was used as the substrate. Therefore, the major role of the His176 in the catalytic mechanism under physiological conditions is not to activate the nucleophilicity of Cys149 but first to facilitate the hydride transfer. These results hypothesized that a phosphorylating GraP-DH possessing a different protein environment competent to increase the nucleophilic character of the essential Cys residue and to favor the hydride transfer in place of His, could be enzymically efficient. This is most likely the case for archaeal Methanothermus fervidus GraP-DH which shares less than 15% amino-acid identity with the bacterial or eukaryotic counterparts. No Cys-/His+ ion pair was detectable. Only one thiolate entity was observed with an apparent pKa of 6.2. This result was confirmed by the fact that none of the mutations of the five invariant His changed the catalytic efficiency.
The coenzyme-bound form of human skeletal muscle d-glyceraldehyde-3-phosphate dehydrogenase has been shown to crystallize in the space group C2 and not C2221 as previously reported. The unit cell contains two tetrameric molecules with the dimer of molecular weight 72,000 as the crystallographic asymmetric unit. The recorded X-ray intensity distribution clearly indicates the presence of non-crystallographic 2-fold axes perpendicular to the crystallographic 2-fold axis showing that the subunits are arranged with near perfect 222 symmetry.Isomorphous derivatives of the enzyme have been prepared and the heavy atom positions defined in complete agreement with the C2 space group assignment. Further confirmation that the space group is C2 and not C2221 comes from the 3.5 Å resolution electron density map of the human enzyme, which appears almost identical to that of the lobster holo-enzyme where no such space group ambiguity exists.
Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
The structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus has been crystallographically refined at 1.8 A resolution using restrained least-squares refinement methods. The final crystallographic R-factor for 93,120 reflexions with F greater than 3 sigma (F) is 0.177. The asymmetric unit of the crystal contains a complete tetramer, the final model of which incorporates a total of 10,272 unique protein and coenzyme atoms together with 677 bound solvent molecules. The structure has been analysed with respect to molecular symmetry, intersubunit contacts, coenzyme binding and active site geometry. The refined model shows the four independent subunits to be remarkable similar apart from local deviations due to intermolecular contacts within the crystal lattice. A number of features are revealed that had previously been misinterpreted from an earlier 2.7 A electron density map. Arginine at position 195 (previously thought to be a glycine) contributes to the formation of the anion binding sites in the active site pocket, which are involved in binding of the substrate and inorganic phosphates during catalysis. This residue seems to be structurally equivalent to the conserved Arg194 in the enzyme from other sources. In the crystal both of the anion binding sites are occupied by sulphate ions. The ND atom of the catalytically important His176 is hydrogen-bonded to the main-chain carbonyl oxygen of Ser177, thus fixing the plane of the histidine imidazole ring and preventing rotation. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. A significant number of buried water molecules have been found that play an important role in the structural integrity of the molecule.
Single crystal X-ray data were collected on film for the holoenzyme of lobster d-glyceraldehyde-3-phosphate dehydrogenase to 3·0 Å resolution. Films of potassium tetraiodomercurate, K2HgI4, comprising a complete low resolution set, with some additional high resolution terms, were given to us by Drs H. C. Watson and L. J. Banaszak. A 3·0 Å high resolution data set was collected of a p-chloromercuri-phenylsulfonate derivative. All these films were processed on a computer controlled Optronics film scanner. The K2HgI4 derivative difference Patterson was initially interpreted in terms of four single sites, one for each polypeptide chain, consistent with the previously determined molecular 222 symmetry. Single isomorphous replacement phases were then sufficient to identify other heavy atom sites. Least-squares refined parameters were used to give multiple isomorphous replacement phases at low resolution, and single isomorphous replacement phases at high resolution. The resultant electron density map was oriented along the molecular 2-fold axes and then averaged over all four equivalent subunits. This process produced a much improved electron density map, which could easily be interpreted in terms of a single polypeptide chain per subunit consistent with the known amino acid sequence. The use of non-crystallographic symmetry to improve the electron density map is equivalent to the molecular replacement method. A comparison is also made with other dehydrogenases.
We have performed a 14-month, prospective, randomized, double-blind, placebo-controlled study to evaluate the effect of deprenyl and levodopa/carbidopa (Sinemet) on the progression of signs and symptoms in patients with mild Parkinson's disease (PD). One hundred one untreated PD patients were randomly assigned to one of the following four treatment groups: Group I, deprenyl + Sinemet; Group II, placebo-deprenyl + Sinemet; Group III, deprenyl + bromocriptine; and Group IV, placebo-deprenyl + bromocriptine. The final visit was performed at 14 months, i.e., 2 months after withdrawal of deprenyl or its placebo and 7 days after withdrawal of Sinemet or bromocriptine. Deterioration in Unified Parkinson's Disease Rating Score (UPDRS) between untreated baseline and final visits was used as an index of disease progression. Placebo-treated patients deteriorated by 5.8 +/- 1.4 points, while deprenyl-treated patients deteriorated by 0.4 +/- 1.3 points (p < 0.001). This effect was sufficiently powerful that a significant deprenyl effect could be detected in the subgroup of 41 patients randomized to Sinemet (p < 0.01) as well as in the 23 patients who completed a 14-day washout of Sinemet or bromocriptine (p < 0.05). No difference in the extent of deterioration was detected in patients randomized to Sinemet versus bromocriptine. This study demonstrates that deprenyl attenuates deterioration in UPDRS score in patients with early PD. These findings are not readily explained by the drug's symptomatic effects and are consistent with the hypothesis that deprenyl has a neuroprotective effect.
The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the trypanosomatid parasite Leishmania mexicana has been determined by X-ray crystallography. The protein crystallizes in space group P2(1)2(1)2(1) with unit cell parameters a = 99.0 A, b = 126.5 A, and c = 138.9 A. There is one 156,000 Da protein tetramer per asymmetric unit. The model of the protein with bound NAD+s and phosphates has been refined against 86% complete data from 10.0 to 2.8 A to a crystallographic Rfactor of 0.198. Density modification by noncrystallographic symmetry averaging was used during model building. The final model of the L. mexicana GAPDH tetramer shows small deviations of less than 0.5 degrees from ideal 222 molecular symmetry. The structure of L. mexicana GAPDH is very similar to that of glycosomal GAPDH from the related trypanosomatid Trypanosoma brucei. A significant structural difference between L. mexicana GAPDH and most previously determined GAPDH structures occurs in a loop region located at the active site. This unusual loop conformation in L. mexicana GAPDH occludes the inorganic phosphate binding site which has been seen in previous GAPDH structures. A new inorganic phosphate position is observed in the L. mexicana GAPDH structure. Model building studies indicate that this new anion binding site is well situated for nucleophilic attack of the inorganic phosphate on the thioester intermediate in the GAPDH-catalyzed reaction. Since crystals of L. mexicana GAPDH can be grown reproducibly and diffract much better than those of T. brucei GAPDH, L. mexicana GAPDH will be used as a basis for structure-based drug design targeted against trypanosomatid GAPDHs.
Fluoxetine and L-deprenyl were prescribed concurrently in a 19-year-old female with Huntington's disease. The patient showed significant affective, behavioral, and motoric improvements and there was no adverse effect resulting from this combination. Concomitant use of L-deprenyl in low doses and fluoxetine may be safe and beneficial in certain clinical situations, although further study is needed.
Within the framework of a project aimed at the structure-based design of drugs for use against sleeping sickness, selective inhibitors were designed, synthesised and tested. The target protein was glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the adenosine part of the NAD cofactor was chosen as lead. After one design cycle and exploiting the selectivity cleft in trypanosomal GAPDH near the C2 of the adenosine ribose, a selective inhibitor, 2'-deoxy-2'-(3-methoxybenzamido)adenosine, was obtained. This compound inhibits human GAPDH only marginally, whereas the enzymes from Trypanosoma brucei and Leishmania mexicana are inhibited by 50% at 2.2 and 0.3 mM, respectively. Moreover, the inhibition of the parasite enzyme is 45-fold (T. brucei) or 170-fold (L. mexicana) greater with this substituted analogue than that produced with adenosine.
Ischemia-induced delayed neuronal death can be mediated by apoptosis, and (-)deprenyl has been shown to block apoptosis in dopaminergic and cholinergic neurons. This study has investigated whether (-)deprenyl can prevent delayed neuronal death of hippocampal pyramidal cells. Rats were subjected to unilateral hypoxia-ischemia and treated with (-)deprenyl (0.25 mg/kg, s.c.) or saline daily. After sacrifice the left and right hippocampi were examined histologically. Unilateral delayed neuronal death was seen in the CA1, CA3 and CA4 fields up to 14 days after the ischemia. After 14 days' treatment with (-)deprenyl there was 66%, 91% and 96% reduction in delayed neuronal death in the CA1, CA3 and CA4 fields, respectively. (-)Deprenyl was effective when given at the onset or after ischemia, but not when given 2 h before ischemia. The reduction in ischemia-induced delayed neuronal death is consistent with an anti-apoptotic mechanism of (-)deprenyl.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels increase in particulate fractions in association with cell death in HEK293 cells, S49 cells, primary thymocytes, PC12 cells, and primary cerebral cortical neuronal cultures. Subcellular fractionation and immunocytochemistry reveal that this increase primarily reflects nuclear translocation. Nuclear GAPDH is tightly bound, resisting extraction by DNase or salt treatment. Treating primary thymocytes, PC12 cells, and primary cortical neurons with antisense but not sense oligonucleotides to GAPDH prevents cell death. Because cell-death-associated nuclear translocation of GAPDH and antisense protection occur in multiple neuronal and nonneuronal systems, we propose that GAPDH is a general mediator of cell death and uses nuclear translocation as a signaling mechanism.
The structure of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from glycosomes of the parasite Trypanosoma cruzi, causative agent of Chagas' disease, is reported. The final model at 2.8 A includes the bound cofactor NAD+ and 90 water molecules per monomer and resulted in an Rfactor of 20.1%, Rfree = 22.3%, with good geometry indicators. The structure has no ions bound at the active site resulting in a large change in the side chain conformation of Arg249 which as a consequence forms a salt bridge to Asp210 in the present structure. We propose that this conformational change could be important for the reaction mechanism and possibly a common feature of many GAPDH structures. Comparison with the human enzyme indicates that interfering with this salt bridge could be a new approach to specific inhibitor design, as the equivalent to Asp210 is a leucine in the mammalian enzymes.
The crystal structures of gyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Escherichia coli have been determined in three different enzymatic states, NAD(+)-free, NAD(+)-bound, and hemiacetal intermediate. The NAD(+)-free structure reported here has been determined from monoclinic and tetragonal crystal forms. The conformational changes in GAPDH induced by cofactor binding are limited to the residues that bind the adenine moiety of NAD(+). Glyceraldehyde 3-phosphate (GAP), the substrate of GAPDH, binds to the enzyme with its C3 phosphate in a hydrophilic pocket, called the "new P(i)" site, which is different from the originally proposed binding site for inorganic phosphate. This observed location of the C3 phosphate is consistent with the flip-flop model proposed for the enzyme mechanism [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. Via incorporation of the new P(i) site in this model, it is now proposed that the C3 phosphate of GAP initially binds at the new P(i) site and then flips to the P(s) site before hydride transfer. A superposition of NAD(+)-bound and hemiacetal intermediate structures reveals an interaction between the hydroxyl oxygen at the hemiacetal C1 of GAP and the nicotinamide ring. This finding suggests that the cofactor NAD(+) may stabilize the transition state oxyanion of the hemiacetal intermediate in support of the flip-flop model for GAP binding.
The properties of the active center of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are considered with emphasis on the structure of anion-binding sites and their role in catalysis. The results of studies on the molecular mechanism of the effect of NAD+ on the enzyme conformation are discussed. Experimental evidence is presented supporting the idea that negative cooperativity of NAD+ binding and half-of-the-sites reactivity exhibited by GAPDH are generated by different mechanisms. Data obtained with rabbit muscle and Escherichia coli GAPDH point to preexisting asymmetry in these tetramers. Structural determinants that can control the transition of the tetramer from the symmetric to the asymmetric state were found.
The structure of the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) from Trypanosoma cruzi complexed with chalepin, a natural product from Pilocarpus spicatus, has been determined by X-ray crystallography to 1.95 A resolution. The structure is in the apo form without cofactors in the subunits of the tetrameric gGAPDH in the asymmetric unit. Unequivocal density corresponding to the inhibitor was clearly identified in one monomer. The final refined model of the complex shows extensive conformational changes when compared with the native structure. The mode of binding of chalepin to gGAPDH and its implications for inhibitor design are discussed.
Many of today's medical illnesses can be attributed directly or indirectly to problems with apoptosis--a programmed cell-suicide mechanism. Disorders in which defective regulation of apoptosis contributes to disease pathogenesis or progression can involve either cell accumulation, in which cell eradication or cell turnover is impaired, or cell loss, in which the cell-suicide programme is inappropriately triggered. Identification of the genes and gene products that are responsible for apoptosis, together with emerging information about the mechanisms of action and structures of apoptotic regulatory and effector proteins, has laid a foundation for the discovery of drugs, some of which are now undergoing evaluation in human clinical trials.
Differences between host and parasite energy metabolism are eagerly sought after as potential targets for antiparasite chemotherapy. In Kinetoplastia, the first seven steps of glycolysis are compartmented inside glycosomes, organelles that are related to the peroxisomes of higher eukaryotes. This arrangement is unique in the living world. In this review, Christine Clayton and Paul Michels discuss the implications of this unusual metabolic compartmentation for the regulation of trypanosome energy metabolism, and describe how an adequate supply of energy is maintained in different species and life cycle stages.
Homotetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus can be described as a dimer of dimers with three non-equivalent P, R, and Q interfaces. In our previous study, negative cooperativity in NAD binding to wild-type GAPDH was interpreted according to the induced-fit model in terms of two independent dimers with two interacting binding sites in each dimer. Two dimeric mutant GAPDHs, i.e. Y46G/S48G and D186G/E276G, were shown to exhibit positive cooperativity in NAD binding. Based on the molecular modeling of the substitutions and the fact that the most extensive inter-subunit interactions are formed across the P-axis interface of the tetramer, it was postulated that both dimeric mutant GAPDHs were of O-P type. Therefore, the P-axis interface was assumed to play a major role in causing cooperativity in NAD binding.Here, two other mutant GAPDHs, Y46G/R52G and D282G, have been studied. Using small angle X-ray scattering, the dimeric form of the D282G mutant GAPDH is shown to be of O-R type whereas both dimeric mutant GAPDHs Y46G/R52G and Y46G/S48G are of O-P type. Similarly to dimeric Y46G/S48G mutant GAPDH, the dimeric Y46G/R52G mutant GAPDH exhibits positive cooperativity in NAD binding. On the other hand, no significant cooperativity in NAD binding to the dimeric form of the D282G mutant GAPDH is observed, whereas its tetrameric counterpart exhibits negative cooperativity, similarly to the wild-type enzyme. Altogether, the results support the view that the P-axis interface is essential in causing cooperativity in NAD binding by transmitting the structural information induced upon cofactor binding from one subunit to the other one within O-P/Q-R dimers in contrast to the R-axis interface, which does not transmit structural information within O-R/Q-P dimers. The absence of activity of O-P and O-R dimer GAPDHs is the consequence of a pertubation of the conformation of the active site, at least of the nicotinamide subsite, as evidenced by the absence of an ion pair between catalytic residues C149 and H176 and the greater accessibility of C149 to a thiol kinetic probe.