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L-Lactate oxidase

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... The three-dimensional structure of GO from spinach has been elucidated by X-ray crystallography (Lindqvist and Branden, 1989) and has been refined to 0.2 nm resolution (Lindqvist, 1989). Prior to these structural studies, a general hypothesis for the catalytic mechanism of FMN oxidases had been postulated, based upon the findings of spectroscopic and kinetic analysis of lactate oxidase (Ghisla and Massey, 1991). ...
... These findings are similar to those reported here for human GO and suggest that the positively charged Arg residue is critical for catalysis. This positive charge is thought to be important for the stabilisation of a highly negatively charged intermediate during catalysis, consistent with the so-called carbanion mechanism that has been postulated for flavoenzyme catalysis (Ghisla and Massey, 1991). ...
Thesis
Glycolate oxidase (GO) is a peroxisomal flavoenzyme which catalyses the oxidation of short chain a-hydroxy acids, notably glycolate. The reaction product, glyoxylate, is an oxalate precursor and GO is thus of potential interest for its role in the pathogenesis of the primary hyperoxalurias. The project aims were to identify human GO, characterise the kinetics and substrate specificity of the enzyme and establish methods for the analysis of relevant metabolic pathways in vitro. The gene for human GO was cloned from liver and expressed in bacterial cells. The cDNA is 1128 bp in length and has a 1113 bp open reading frame encoding a 372 amino acid protein. The genomic sequence comprises eight exons and spans —57 kb of chromosome 20p12. Recombinant human GO protein shares 53% and 89% sequence similarity to GO from spinach and rat respectively, shows a-hydroxy acid oxidase activity in vitro and has been purified to homogeneity. Polyclonal anti-GO antibody detects a band of 43 kDa in human liver and, consistent with northern blot analysis, expression is not detected in other tissues including kidney and leucocytes. Kinetic analysis with a range of a-hydroxy acids indicates GO has highest affinity for glycolate as substrate (Km = 0.54 mM) and 10 fold less affinity for glyoxylate (Km = 5.1 mM). Site directed mutagenesis of active site residues demonstrates the importance of chain length for substrate affinity. Thus mutation of a Trp residue, conserved between spinach and human GO to a less bulky amino acid, permits the catalysis of longer chain length a-hydroxy acids. HPLC methods were developed for the separation and quantitation of glyoxylate, hydroxypyruvate and pyruvate, enabling analysis of metabolites produced by GO and neighboring enzymes in the metabolic pathway. These assays will be invaluable for future studies in which the pathways of glyoxylate metabolism are constructed in vitro.
... Since the determination of the three-dimensional structure of spinach glycolate oxidase by X-ray crystallography (Lindqvist and BrandCn, 1989) mechanistic models have been put forward to describe the involvement of the active-site amino acid residues in substrate binding and the catalytic steps (Ghisla and Massey, 1991;Lederer and Mathews, 1987;Lindqvist and BrandCn, 1989). One of the two tyrosine residues in the active site, Tyr24, has been suggested to be involved in binding of the substrate by way of hydrogen-bond formation between the hydroxyl group and the carboxylate group of the substrate molecule. ...
... For example, the reduction of the flavin in lactate monooxygenase is about three-times slower in [Y44F] lactate monooxygenase (Mueh et al., 1994), a value very similar to that found with [Y24F]glycolate oxidase (1.6-fold slower). Earlier proposals by Lederer and Mathews (1987) and Ghisla and Massey (1991) also included stabilization of a transiently formed carbanion through delocalization of the negative charge. Clearly, this proposal cannot be supported by our results. ...
Article
Tyr24 and Trp108 are located in the active site of spinach glycolate oxidase. To elucidate their function in substrate binding and catalysis, they were replaced by phenylalanine and serine, respectively. The [Y24F] glycolate oxidase mutant enzyme showed a tenfold higher Km value for glycolate. l-lactate and dl-2-hydroxybutyrate also showed higher Km values, however, the substrate specificity was unchanged as compared to the wild-type enzyme (Km increases in the order glycolate < dl-2–hydroxybutyrate < l-lactate < l-mandelate). The turnover number and the rate of reduction, found to be rate limiting in catalysis, were only slightly affected by the deletion of the hydroxyl group. These findings suggest that Tyr24 is mostly involved in substrate binding. The spectral features of the [Y24F]glycolate oxidase suggest that a fraction (50–80%) of the protein bears a flavin N(5) adduct instead of the oxidized cofactor. Crystals obtained from the isolated [Y24F] glycolate oxidase mutant protein allowed the determination of the three-dimensional structure. Although the structure was low resolution (0.3 nm), it is evident that the structure determined is that of the N(5) adduct species. In addition to the lacking hydroxyl group of Tyr24, we also observed movements of the amino acid side chains of Argl64 and Trp108, the latter replacing a water molecule in the substrate-binding pocket. Other features predominantly found in the class of flavoprotein oxidases, such as stabilization of the covalent N(5)-sulfite adduct and of the paraquinoid form of 8-mercapto-FMN, were found to be conserved. [W108S]Glycolate oxidase, in contrast, showed dramatic effects on both the Km of substrates as well as on the turnover number. The Km for glycolate was increased some hundred fold and the turnover number was decreased 500–fold. In addition, it was found that the higher homologs of glycolate, l-lactate and dl-2-hydroxybutyrate had turnover numbers similar to those found with the wild-type enzyme, although the Km values also increased dramatically. These results indicate that Trp108 is of major importance in catalysis and that this residue is involved in determining the substrate specificity of glycolate oxidase.
... A feature of many flavin-dependent oxidases is that they form relatively stable reduced enzyme/product complexes, which often have characteristic charge transfer absorptions and can be detected spectrophotometrically [19]. For this reason, formation of the fully reduced uncomplexed species is often seen to follow a biphasic course. ...
Article
The kinetic properties of two cholesterol oxidases, one from Brevibacterium sterolicum (BCO) the other from Streptomyces hygroscopicus (SCO) were investigated. BCO works via a ping-pong mechanism, whereas the catalytic pathway of SCO is sequential. The turnover numbers at infinite cholesterol and oxygen concentrations are 202 s−1 and 105 s−1 for SCO and BCO, respectively. The rates of flavin reduction extrapolated to saturating substrate concentration, under anaerobic conditions, are 235 s−1 for BCO and 232 s−1 for SCO (in the presence of 1% Thesit and 10% 2-propanol). With reduced SCO the rate of Δ5-6→Δ4-5 isomerization of the intermediate 5-cholesten-3-one to final product is slow (0.3 s−1). With oxidized SCO and BCO the rate of isomerization is much faster (≈ 300 s−1), thus it is not rate-limiting for catalysis. The kinetic behaviour of both reduced COs towards oxygen is unusual in that they exhibit apparent saturation with increasing oxygen concentrations (extrapolated rates ≈ 250 s−1 and 1.3 s−1, for BCO and SCO, respectively): too slow to account for catalysis. For BCO the kinetic data are compatible with a step preceding the reaction with oxygen, involving interconversion of reactive and nonreactive forms of the enzyme. We suggest that the presence of micelles in the reaction medium, due to the necessary presence of detergents to solubilize the substrate, influence the availability or reactivity of oxygen towards the enzyme. The rate of re-oxidation of SCO in the presence of product is also too slow to account for catalysis, probably due to the impossibility of producing quantitatively the reduced enzyme–product complexes.
... Their crystal structures show a high degree of similarity, both in the b 8 a 8 fold and around the flavin [8][9][10]. Family members stabilize the anionic semiquinone at physiological pH [11][12][13][14][15]. This has been demonstrated to be the case for flavocytochrome b 2 and its FDH domain [11,12] and should be the case for LCHAO, which is an isozyme of spinach glycolate oxidase [6,16]. ...
Article
The reactions of the flavin semiquinone generated by laser-induced stepwise two-photon excitation of reduced flavin have been studied previously (El Hanine-Lmoumene C & Lindqvist L. (1997) Photochem Photobiol 66, 591-595) using time-resolved spectroscopy. In the present work, we have used the same experimental procedure to study the flavin semiquinone in rat kidney long-chain hydroxy acid oxidase and in the flavodehydrogenase domain of flavocytochrome b(2) FDH, two homologous flavoproteins belonging to the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes. For both proteins, pulsed laser irradiation at 355 nm of the reduced enzyme generated initially the neutral semiquinone, which has rarely been observed previously for these enzymes, and hydrated electron. The radical evolved with time to the anionic semiquinone that is known to be stabilized by these enzymes at physiological pH. The deprotonation kinetics were biphasic, with durations of 1-5 micros and tens of microseconds, respectively. The fast phase rate increased with pH and Tris buffer concentration. However, this increase was about 10-fold less pronounced than that reported for the neutral semiquinone free in aqueous solution. pK(a) values close to that of the free flavin semiquinone were obtained from the transient protolytic equilibrium at the end of the fast phase. The second slow deprotonation phase may reflect a conformational relaxation in the flavoprotein, from the fully reduced to the semiquinone state. The anionic semiquinone is known to be an intermediate in the flavocytochrome b(2) catalytic cycle. In light of published kinetic studies, our results indicate that deprotonation of the flavin radical is not rate-limiting for the intramolecular electron transfer processes in this protein.
... Several studies on Fcb2 have focused on the mechanism of lactate dehydrogenation, catalyzed by its flavodehydrogenase domain, which is the structural and mechanistic prototype of a family of l-a-hydroxy acidoxidizing enzymes [4][5][6][7][8]. This class of enzymes includes, as well-characterized members, glycolate oxidase [9], l-lactate monoxygenase (LMO) [10,11], l-lactate oxidase [10], mandelate dehydrogenase [12] and long-chain a-hydroxy acid oxidase [13]. Crystal structures are known for all these enzymes, except LMO. ...
Article
First principles molecular dynamics studies on active-site models of flavocytochrome b2 (L-lactate : cytochrome c oxidoreductase, Fcb2), in complex with the substrate, were carried out for the first time to contribute towards establishing the mechanism of the enzyme-catalyzed L-lactate oxidation reaction, a still-debated issue. In the calculated enzyme-substrate model complex, the L-lactate alpha-OH hydrogen is hydrogen bonded to the active-site base H373 Nepsilon, whereas the Halpha is directed towards flavin N5, suggesting that the reaction is initiated by alpha-OH proton abstraction. Starting from this structure, simulation of L-lactate oxidation led to formation of the reduced enzyme-pyruvate complex by transfer of a hydride from lactate to flavin mononucleotide, without intermediates, but with alpha-OH proton abstraction preceding Halpha transfer and a calculated free energy barrier (12.1 kcal mol(-1)) consistent with that determined experimentally (13.5 kcal mol(-1)). Simulation results also revealed features that are of relevance to the understanding of catalysis in Fcb2 homologs and in a number of flavoenzymes. Namely, they highlighted the role of: (a) the flavin mononucleotide-ribityl chain 2'OH group in maintaining the conserved K349 in a geometry favoring flavin reduction; (b) an active site water molecule belonging to a S371-Wat-D282-H373 hydrogen-bonded chain, conserved in the structures of Fcb2 family members, which modulates the reactivity of the key catalytic histidine; and (c) the flavin C4a-C10a locus in facilitating proton transfer from the substrate to the active-site base, favoring the initial step of the lactate dehydrogenation reaction.
Article
Amino acid misincorporation during protein synthesis occurs naturally at a low level. Protein sequence errors, depending on the level and the nature of the misincorporation, can have various consequences. When site-directed mutagenesis is used as a tool for understanding the role of a side chain in enzyme catalysis, misincorporation in a variant with intrinsically low activity may lead to misinterpretations concerning the enzyme mechanism. We report here one more example of such a problem, dealing with flavocytochrome b2 (Fcb2), a lactate dehydrogenase, member of a family of FMN-dependent L-2-hydroxy acid oxidizing enzymes. Two papers have described the properties of the Fcb2 catalytic base H373Q variant, each one using a different expression system with the same base change for the mutation. The two papers found similar apparent kinetic parameters. But the first one demonstrated the existence of a low level of histidine misincorporation, which led to an important correction of the variant residual activity (Gaume et al. (1995) Biochimie, 77, 621). The second paper did not investigate the possibility of a misincorporation (Tsai et al. (2007) Biochemistry, 46, 7844). The two papers had different mechanistic conclusions. We show here that in this case the misincorporation does not depend on the expression system. We bring the proof that Tsai et al. (2007) were led to an erroneous mechanistic conclusion for having missed the phenomenon as well as for having misinterpreted the crystal structure of the variant. This work is another illustration of the caution one should exercise when characterizing enzyme variants with low activity.
Article
L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr215 in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr215, effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr215 can thus lead to a kinetic bottleneck in product release.
Article
Flavins are one of the most chemically diverse prosthetic groups in biochemistry. Flavins can react in one- or two-electron redox reactions as well as form covalent adducts that can also perform reactions. The protein environment of an enzyme controls the reactions available, enhancing one or two modes of reactivity while suppressing competing reactions. Flavoenzymes can oxidize carbon–heteroatom bonds, activated carbon–carbon bonds, thiols, and metal electron donors. There are also many flavoenzymes that catalyze the production of such compounds by reduction reactions. Additionally, O2 reacts with reduced flavins, forming H2O2 or flavin hydroperoxide, which is used for hydroxylations. The examples considered in this chapter demonstrate the wide range of chemistry available to flavins and the large number of enzyme catalysts possible by the combination of various reactions. This chemical versatility makes flavins central participants in all biological processes.
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Software for the automatic non-linear least squares fit of chronoamperometric responses corresponding to sandwich-type amperometric biosensors has been developed. The so-called Simplex algorithm computes a minimum value for the difference between experimental and theoretical data. The latter consider a numerical model based on a ping-pong reaction mechanism corresponding to an oxidase enzyme that has been immobilized between diffusion membranes.The results obtained from the simulation of a first-generation lactate biosensor in presence of 0.1 mM substrate indicate that the concentration of O2 would decrease only 0.1% with regards to its bulk value. Besides, the concentration of this natural mediator would remain practically unchanged during a typical calibration curve. This is because the rather high diffusion coefficient of O2 and its regeneration at the electrode surface minimize the concentration changes of this species. In addition, it was found that the thicknesses of polycarbonate membranes and the enzymatic matrix have average values of 13 μm and 20 μm, respectively. However, these membranes might exhibit smaller thickness depending on the time provided for the crosslinking reaction. In this regard, if this reaction is slow enough, the enzymatic matrix would be able to diffuse through the pores of polycarbonate membranes and they will appear to be thinner than expected. This effect may compromise the response-time and the reproducibility of this kind of biosensors.
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The ways in which various flavoproteins modulate the reactivity of the reduced flavin with molecular oxygen constitute a major unsolved problem in the field. The contributions of redox potentials, both of the flavin and the O2/O2− couple, their dependence on the protein environment surrounding the flavin, and the possibility of control through multi-step access of oxygen to the reduced flavin are discussed.
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Full-text available
Oxalate and malonate have been found to bind to the flavoenzyme, lactate oxidase, in a two-step equilibrium process. The dissociation constant for the first step is of the order of lo power -2 M, and is independent of pH. The second step equilibrium is on the other hand directly proportional to [H+] due to the second forward step, which is a second order reaction involving proton uptake. The reverse of this step is a first order reaction involving proton release, and consequently is independent of pH. Simultaneous measurement of proton uptake and formation of the enzyme. oxalate complex show that the rate of proton uptake is coincident with the large spectral changes occurring on binding, with the stoichiometry of one proton uptake per equivalent of enzyme-bound flavin. These results give strong support to the concept that oxalate and malonate are transition state analogs of a carbanion form of the substrate, formed in an early step in the enzyme reaction mechanism. The enzyme base responsible for abstraction of the proton from the (α-carbon of the substrate is postulated to be the same one which is protonated (from the medium) in the second step of binding of oxalate and malonate, to form the transition state equivalent to that occurring with the substrate carbanion in catalysis. The pK of this group has been determined as 4.7, and is shifted to 8.2 and 9.8 on binding with malonate and oxalate, respectively. These pK shifts represent stabilizations of ~5 and ~7 kcal/mole of the enzyme’inhibitor complexes. These findings offer strong support for the intermediate involvement of a substrate carbanion in catalysis.
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Evidence from product studies and radical trapping experiments indicates that the mechanism of the oxidation of the carbonion of methyl 2-methoxy-2-phenylacetate by a model flavin compound in basic methanol is free radical in nature.
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The stoichiometry of flavin photoreduction was studied by flash photolysis, monitoring the conversion of photoexcited flavin to one-electron and two-electron reduction products and following their fate. The reactivity of the photoexcited states of flavin in one-electron transfer reactions was determined, the reasons for differences between them are discussed and the mode of radical decay was investigated in dependence of the photosubstrate structure. The nature of the two-electron transferring photosubstrate was revealed in order to define the range of the two reduction pathways.
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The time courses for the appearance of lumiflavin-3-acetate (Fl ox) on reaction of 1,5-dihydrolumiflavin-3-acetate (FlH 2) and its N(1) anion (FlH -) with pyruvamide, ethyl pyruvate, pyruvic acid, and pyruvate anion are quantitatively explained by the reactions of Scheme I. In competitive reactions FlH 2 and FlH - (FlH 2T) react with the carbonyl substrate to yield Fl ox and the N(5)-carbinolamine (CA) which goes on to the corresponding imine (Im). This initial process (Fl ox ← FlH 2T ⇄ CA T ⇄ Im T) may be characterized by an initial burst of Fl ox production dependent upon pH and carbonyl substrate. Following this initial phase, Fl ox continues to be formed by two pathways. Return of CA to FlH 2T contributes but the principal production of Fl ox occurs via the comproportionation of Fl ox with CA [which is an N(5)-alkyl-1,5-dihydroflavin]. Formation of CA and Im during the course of the reaction is supported by their spectral identification (at 355-360 and 500 nm, respectively) and by the quantitative reductive trapping with sodium cyanoborohydride and subsequent conversion of the resultant N-alkyl-1,5-dihydroflavin to its aminium cation radical (λ max ∼585 nm). The concentration of FlH 2T remaining in solution, following the initial burst reaction, has been determined via its rapid conversion to Fl ox by addition of CH 2O or additional substrate. An extended kinetic study was deemed feasible only for pyruvic acid and pyruvate due to the competing hydrolysis of ethyl pyruvate and the observed dimerization of pyruvamide during the time of reaction with FlH 2T. The time courses for formation of Fl ox from the reaction of pyruvic acid + pyruvate with FlH 2T have been simulated by analog computations employing the differential expressions for the reactions of Scheme II. The rate constants obtained from the analog simulation have been fit to log k rate vs. pH profiles and k rate vs. substrate concentration plots. For ethyl pyruvate and pyruvamide, selective analog simulation of the time courses for Fl ox production suffice to support the mechanism of Scheme I. The direct reaction of ethyl pyruvate with FlH 2T to yield ethyl lactate and Fl ox is first order in reactants. At pH 1 the reduction of pyruvic acid + pyruvate is first order in [pyruvic acid + pyruvate] and [H 3O +], while at pH 3.3 the reaction is second order in [pyruvic acid + pyruvate] and is [H 3O +] independent. Mechanisms involving le - transfer from 1,5-dihydroflavin species to the carbonyl group of the substrate which involve proton transfer to substrate carbonyl oxygen or its derived anion radical are considered. The reasonableness of radical pair intermediates (e.g., FlH-· T·C-O - and FlH· T·C-OH) along the reaction path is established from the observation that the computed standard free energies of formation for these species in the pH range of investigation are less positive by 9 to 14 kcal M -1 than ΔG ‡expt. At alkaline pH values the standard free energy for pyruvate + FlH - → lactate + Fl ox is positive, and it has been shown that lactamide at pH 11.6 and lactate at pH 10 reduces Fl ox to FlH -. The kinetics and mechanism for CA and Im formation and the comproportionation of CA and Fl ox are discussed.
Article
The three-dimensional structure of flavocytochrome b2 (L-lactate dehydrogenase) from bakers' yeast (Saccharomyces cerevisiae) has recently been solved at 0.24-nm resolution [Mathews & Xia (1987) in Flavins and flavoproteins, Walter de Gruyter, Berlin, pp. 123–131]. We have used this structural information to investigate the roles of particular amino acid residues likely to be involved in the oxidation of L-lactate by kinetic analysis of mutant enzymes generated by site-directed mutagenesis of the isolated gene. The hydroxyl group of Tyr254 was expected to be important for the abstraction of the hydroxyl proton of L-lactate in the oxidation to pyruvate. Replacement of this tyrosine by phenylalanine reduced kcat from 190 ± 3 s−1 (25°C, pH 7.5) to 4.3 ± 0.1 s−1. This substitution had, however, no discernable effect on Km for lactate (0.54 ± 0.03 mM for the mutant compared with 0.49 ± 0.03 mM for the wild-type enzyme). Arg376 was expected to be essential for productive binding and orientation of L-lactate. Replacing Arg376 with lysine abolished all detectable activity. A total loss of enzymic activity was also observed when Lys349, thought likely to stabilize the anionic form of the flavin hydroquinone, was replaced by arginine. An amino acid residue replacement at a distance from the active site, Ala306 to serine, had a minor but significant effect on kcat (reduced from 190 s−1 to 160 s−1) and Km (increased from 0.49 mM to 0.83 mM) presumably arising from small conformational effects. The implications of these results are discussed in relation to the mechanism of l-lactate oxidation.