Binding Of L-lactate Research Articles

Dynamic interactions in the l-lactate oxidase active site facilitate substrate binding at pH4.5

The crystal structure of l-lactate oxidase in complex with l-lactate was solved at a 1.33 Å resolution. The electron density of the bound l-lactate was clearly shown and comparisons of the free form and substrate bound complexes demonstrated that l-lactate was bound to the FMN and an additional active site within the enzyme complex. l-lactate interacted with the related side chains, which play an important role in enzymatic catalysis and especially the coupled movement of H265 and D174, which may be essential to activity. These observations not only reveal the enzymatic mechanism for l-lactate binding but also demonstrate the dynamic motion of these enzyme structures in response to substrate binding and enzymatic reaction progression.

Speeding up the product release: a second‐sphere contribution from Tyr191 to the reactivity of l‐lactate oxidase revealed in crystallographic and kinetic studies of site‐directed variants

Among α-hydroxy acid-oxidizing flavoenzymes l-lactate oxidase (LOX) is unique in featuring a second-sphere tyrosine (Tyr191 in Aerococcus viridans LOX; avLOX) at the binding site for the substrate's carboxylate group. Y191F, Y191L and Y191A variants of avLOX were constructed to affect a hydrogen-bond network connecting Tyr191 to the carboxylate of the bound ligand via the conserved Tyr40 and to examine consequent effects on enzymatic reactivity. Kinetic studies at 20 °C and pH 6.5 revealed that release of pyruvate product was decreased 4.7-fold (Y191F), 19-fold (Y191L) and 28-fold (Y191A) compared with wild-type enzyme (~ 141 s(-1)) and thus became mainly rate limiting for l-lactate oxidation by the variants at a steady-state under air-saturated conditions. In the Y191L and the Y191A variants, but not in the Y191F variant, l-lactate binding was also affected strongly by the site-directed substitution. Reduction of the flavin cofactor by l-lactate and its reoxidation by molecular oxygen were, however, comparatively weakly affected by the replacements of Tyr191. Unlike the related lactate monooxygenase, which prevents the fast dissociation of pyruvate to promote its oxidative decarboxylation by H2 O2 into acetate, CO2 and water as final reaction products, all avLOX variants retained their native oxidase activity where catalytic turnover results in the equivalent formation of H2O2. The 1.9 Å crystal structure of the Y191F variant bound with FMN and pyruvate revealed a strictly locally disruptive effect of the site-directed substitution. Product off-rates appear to be dictated by partitioning of residues including Tyr191 from an active-site lid loop into bulk solvent and modulation of the hydrogen bond strength that links Tyr40 with the pyruvate's carboxylate group. Overall, this study emphasizes the possibly high importance of contributions from second-sphere substrate-binding residues to the fine-tuning of reactivity in α-hydroxy acid-oxidizing flavoenzymes, requiring that the catalytic steps of flavin reduction and oxidation are properly timed with the physical step of α-keto acid product release.

MD and QM/MM studies on long-chain L-α-hydroxy acid oxidase: substrate binding features and oxidation mechanism.

Long-chain L-α-hydroxy acid oxidase (LCHAO) is a flavin mononucleotide (FMN)-dependent oxidase that dehydrogenates l-α-hydroxy acids to keto acids. There were two different mechanisms, named as hydride transfer (HT) mechanism and carbanion (CA) mechanism, respectively, proposed about the catalytic process for the FMN-dependent L-α-hydroxy acid oxidases on the basis of biochemical data. However, crystallographic and kinetic studies could not provide enough evidence to prove one of the mechanisms or eliminate the alternative. In the present studies, theoretical computations were carried out to study the molecular mechanism for LCHAO-catalyzed dehydrogenation of L-lactate. Our molecular dynamics (MD) simulations indicated that L-lactate prefers to bind with LCHAO in a hydride transfer mode rather than a carbanion mode. Quantum mechanics/molecular mechanics (QM/MM) calculations were further carried out to obtain the optimized structures of reactants, transition states, and products at the level of ONIOM-EE (B3LYP/6-311++G(d,p)//B3LYP/6-31G(d,p):AMBER). Quantum chemical studies indicated that LCHAO-catalyzed dehydrogenation of L-lactate would be a stepwise catalytic reaction in a hydride transfer mechanism but not a carbanion mechanism. MD simulations, binding free energy calculations, and QM/MM computations were also implemented on the complex between L-lactate and Y129F mutant LCHAO. By comparing the Y129F mutant system with the wild-type system, it was further confirmed that the key residue Tyr129 in the active site of LCHAO would not affect L-lactate's binding to LCHAO but play an important role on the catalytic reaction process through an H-bond interaction.

Differential regulation of hexameric and dodecameric hemocyanin from A. leptodactylus

The oxygen binding properties of hemocyanins are regulated on a short time scale by effectors such as l-lactate, urate and protons, and on longer time scales by expression of the different types of subunits. For Astacus leptodactylus it was shown previously that acclimation to higher temperatures leads to increased levels of a 6-meric hemocyanin species, whereas at lower temperatures the 12-meric form prevails. Here we show that the temperature dependence of the two forms supports the idea, that the maintenance of high affinity towards oxygen is the driving force for the differential expression of these hemocyanins. Furthermore, the two different types of hemocyanin differ not only in the affinity to oxygen, but also with respect to their interaction with l-lactate: while the 12-meric form displays a normal shift in oxygen affinity upon the addition of l-lactate this allosteric regulation is absent in the 6-meric form. Exclusive binding of l-lactate to the 12-meric form was supported by isothermal titration calorimetry. These results indicate that l-lactate binds either at the interface between the two hexamers or at subunit α′ which is responsible for the formation of the 12-mers and is not present in the 6-meric form. Urate has a comparable effect on the oxygen affinity of 6-meric and 12-meric forms and also binds to a similar extent to the oxygenated state as determined by isothermal titration calorimetry. Thus, urate and l-lactate do not seem to share the same binding sites. Interestingly, urate binding sites with no allosteric effect seem to exist, which is unusual. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

The Roles of Two Amino Acid Residues in the Active Site of l-Lactate Monooxygenase: MUTATION OF ARGININE 187 TO METHIONINE AND HISTIDINE 240 TO GLUTAMINE

Lactate monooxygenase (LMO) catalyzes the conversion of L-lactate to acetate, CO(2), and water with the incorporation of molecular oxygen. Arginine 187 of LMO is highly conserved within the family of L-alpha-hydroxyacid oxidizing enzymes (Lê, K. H. D., and Lederer, F. (1991) J. Biol. Chem. 266, 20877-20881). By comparison with the equivalent residue in flavocytochrome b(2) from Saccharomyces cerevisiae (Pike, A. D., Chapman, S. K, Manson, F. D. C,. Reid, G. A. , Gondry, M., and Lederer, F. (1996) in Flavins and Flavoproteins (Stevenson, K. J., Massey, V., and Williams, C. H., Jr., eds) pp. 571-574, University of Calgary Press, Calgary, AB, Canada), arginine 187 might be expected to have an important role in catalytic efficiency and substrate binding in LMO. Histidine 240 is predicted to be close to the substrate binding site of LMO, although it is not conserved within the enzyme family. Arginine 187 has been replaced with methionine (R187M), and histidine 240 has been replaced with glutamine (H240Q). L-Lactate oxidation by R187M is very slow. The binding of L-lactate to the mutant enzyme appears to be very weak, as is the binding of oxalate, a transition state analogue. The binding of pyruvate to the reduced enzyme is also very weak, resulting in complete uncoupling of enzyme turnover, with H(2)O(2) and pyruvate as the final products. In addition, anionic forms of the flavin are unstable. The K(d) for sulfite is increased nearly 400-fold by this mutation. The semiquinone form of R187M is also thermodynamically unstable, although the overall midpoint potential for the two-electron reduction of R187M is only 34 mV lower than for the wild-type enzyme. H240Q more closely resembles the wild-type enzyme. The steady-state activity of H240Q is completely coupled. The k(cat) is similar to that for the wild-type enzyme.

Determination of l-lactate binding stoichiometry and differences in allosteric interactions of structurally distinct homohexamers from Panulirus interruptus hemocyanin

The role of structurally distinct subunits from the hemocyanin of Panulirus interruptus was investigated by the analysis of the oxygen-binding properties of reassembled homohexamers. homohexamers reassembled from subunits a and b exhibited cooperative oxygen binding, whereas subunit c did not. The oxygen affinity of homohexamers from subunit b and c was specifically increased by the addition of l-lactate, whereas that of subunit a was not. Both native hexamers and the homohexamers from subunit b have approximately one oxygen-linked lactate binding site per hexamer.

A model for l-lactate binding to Cancer magister hemocyanin

1. 1. l-Lactate raises the oxygen affinity of Cancer magister hemocyanin. The l-lactate analogs, d-lactate, glycolate and 2-methyl-lactate cause smaller increases in an oxygen binding affinity. Other analogs have no detectable effect. 2. 2. These data suggest that l-lactate binds to the hemocyanin at all four positions around the chiral carbon. 3. 3. The carboxyl and hydroxyl groups are required for activity. The protein can only partially distinguish between the methyl group and hydrogen atom.

Allosteric modulation of Callinectes sapidus hemocyanin by binding of L-lactate.

Hemocyanin of the blue crab Callinectes sapidus has the typical structure of crustacean hemocyanins in that its smallest in vivo structure is a hexamer of subunits each having a molecular mass of approximately 75 000. As found in the blood, Callinectes hemocyanin consists of a mixture of hexamers and dodecamers (typically 1:4). As in other crustacean hemocyanins, the affinity with which oxygen binds to the binuclear copper site has been reported to be very sensitive to pH and to a variety of inorganic allosteric effectors. We report here the interaction of L-lactate, a natural metabolite,with the native hemocyanin and with chromatographically purified hexamers and dodecamers. Under ionic conditions that approximate those found physiologically, the addition of 10 mM L-lactate to native Callinectes hemocyanin substantially increases its oxygen affinity (Δ log P(50) = -0.28). The data from lactate titrations were fit to a theoretical equation,and the best fit was obtained with a lactate dissociation constant of 1.8 mM for the oxy state and 2.2 lactate binding sites for every 6 oxygen binding sites. Independent measurements by ultrafiltration techniques indicated a dissociation constant of 3.2 mM with 2.8 lactate binding sites per 6 oxygen binding sites. The two sets of data clearly indicate that there is less than one lactate binding site per oxygen binding site. The fit to the titration was not improved with the assumption of more than one class of lactate binding site. The hexamers and dodecamers of native Callinectes hemocyanin are not in equilibrium and are stable after separation by gel-filtration chromatography. Polyacrylamide gel electrophoresis of the subunits of the dissociated dodecamers shows five major bands.Two of these bands, which constitute one-sixth of the total dodecameric hemocyanin, do not appear upon gel electrophoresis of dissociated hexamers. The oxygen affinities of the hexameric and dodecameric hemocyanin forms are similar to one another but show differences in their sensitivity to L-lactate.The oxygen affinity of native Callinectes hemocyanin was increased appreciably more by L-lactate than by glycolate,D-lactate, and pyruvate (listed in decreasing order of effectiveness).Propionate, acetate, succinate, D-alanine, and L-alanine were without effect, thus illustrating the selectivity of the L-lactate effect on the hemocyanin. We found the magnitude of the Bohr effect to be unchanged by the addition of L-lactate over the pH range 7.5-8.0. Moreover, there is no significant effect of L-lactate on the aggregation state of the hemocyanin or on its 340-nm copper-oxygen absorption band.The foregoing results are consistent with the role of L-lactate as a specific allosteric effector of Callinectes hemocyanin that acts by preferential binding to a stereospecific site of the oxy hemocyanin.

Use of isotope effects and pH studies to determine the chemical mechanism of Bacillus subtilis L-alanine dehydrogenase.

Analysis of deuterium isotope effects with L-alanine-d4 and L-serine-d3, and of pH profiles with the same substrates, shows that L-alanine is sticky (that is, reacts to give products 1-7 times as fast as it dissociates) while L-serin is not. The pH profiles show the following: (1) NH3 and monoanionic amino acids are the substrates; (2) a cationic acid group on the enzyme (probably lysine) with a pK of 9.0-9.6 in E-NAD, but a pK well above 10 in E-NADH, must be protonated for activity and good binding of inhibitors and is probably important for maintaining the proper conformation of the enzyme; (3) A cationic acid group on the enzyme (probably histidine) with a pK around 7 in both E-NAD and E-NADA must be unprotonated for oxidation of amino acids but protonated for binding and reaction of pyruvate. This latter group is the acid-base catalyst for the chemical reaction. In E-NAD, it is so positioned that it can hydrogen bond to (and thus when protonated enhance the binding of) a D-hydroxy or a carbonyl group of an inhibitor, but its state of protonation does not affect the binding of L-lactate or propionate. In E-NADH, it is so placed that it can hydrogen bond to both D- and L-hydroxy groups, as well as in carbonyl groups. A chemical mechanism is postulated in which the dehydrogenation of L-alanine by NAD to produce iminopyruvate is followed by attack of water from the same side from which the hydride was removed. The catalytic histidine transfers a proton from the attacking water to the amino group of the resulting carbinolamine and then removes a proton from the hydroxyl group of the carbinolamine as ammonia is eliminated to give pyruvate.

Kinetic formulations for the oxidation and the reduction of glyoxylate by lactate dehydrogenase

Chicken liver lactate dehydrogenase ( l-lactate : NAD + oxidoreductase, EC 1.1.1.27) irreversibly catalyses the oxidation of glyoxylate (hydrated form)(I) to oxalate ( pH = 9.6 ) and the reduction of (non-hydrated form) (II) to glycolate ( pH = 7.4 ). (I) attaches to the enzyme in the pyruvate binding site and (II) attaches to the enzyme at the l-lactate binding site. The oxidation of (I) ( pH = 9.6 ) is adapted to the following mechanism: The abortive complexes, E-NADH-I and E-NAD +-II, are responsible for the inhibition by excess substrate in the reduction and oxidation systems, respectively. When lactate dehydrogenase and NAD + are preincubated, E-NAD +NAD + appears and causes inhibition by excess NAD + in the glyoxylate-lactate dehydrogenase-NAD + and l-lactate-lactate dehydrogenase-NAD + systems; the second NAD + molecule attaches to the enzyme at the l-lactate binding site.