Hydride Transfer Step Research Articles

Transition structure for hydride transfer from cyclopropene to azirinium cation

The transition structure for the hydride transfer between the cyclopropene and azirinium cation has been characterized by means of the ab initio method at the 3-21G, 4-31G, 6-31G, 6-31 + + G ∗ basis set levels. There are two transition structures for the exo and endo conformations. The transferring hydrogen is located closer to the acceptor centre, nitrogen atom, than to the donor centre, carbon atom, with an almost bent arrangement. The electronic structure and transition vector are basis set level independent. For the transition structures, the endo is more stable than the exo conformation due to a favourable orbital interaction. An analysis of calculated transition structure associated with the hydride transfer step in different model systems shows that the use of very simple models to represent this step do not alter their nature. The transition structures for hydride transfer step seems to be a robust entity. Furthermore, there is a minimal molecular model with a transition structure and transition vector which describes the essentials of the chemical interconversion step.

On Transition Structures for Hydride Transfer Step: A Theoretical Study of the Reaction Catalyzed by Dihydrofolate Reductase Enzyme

A theoretical study is presented of the catalytic mechanism of dihydrofolate reductase (DHFR) enzyme based upon the characterization of the transition structure (TS) for the hydride transfer step. Analytical gradients at AM1 and PM3 semiempirical levels have been used to characterize the saddle point of index one (SPi-1) on global energy hypersurface for the hydride transfer in the active site of DHFR enzyme. The geometry, stereochemistry, electronic structure, and transition vector (TV) components associated to SPi-1 are qualitatively computational level independent. The TV amplitudes show primary and secondary isotope effects to be strongly coupled. The geometrical arrangement of the TS results in optimal frontier orbital interaction. Comparison of the TS geometry with the X-ray coordinates shows that the TS can be fitted without any stress at the active site. By comparison of the TS for the hydride transfer step in models of related enzymes, the geometries and the components of TV for TSs in hydride transfer steps are transferable and invariant. The results of this study suggest that the primary function of the enzyme is to constrain the reactants in the endo configuration, situating the system in a neighborhood of a SPi-1. This fact is in accordance with Pauling's postulate for enzyme catalysis.

On a possible invariance of a transition structure to the effects produced by ancillary H-bonding molecules: Modeling the effects of Ser-48 in the hydride-transfer step of liver alcohol dehydrogenase

The influence of a hydroxyl group simulating Ser-48 in the hydride-transfer step characteristic of liver alcohol dehydrogenase is studied on the hydride-transfer reaction as modeled by a methanolate anion interacting with a cycle propenyl cation. It is sh

On Transition Structures for Hydride Transfer Step in Enzyme Catalysis. A Comparative Study on Models of Glutathione Reductase Derived from Semiempirical, HF, and DFT Methods

As a model of the chemical reactions that take place in the active site of gluthatione reductase, the nature of the molecular mechanism for the hydride transfer step has been characterized by means of accurate quantum chemical characterizations of transition structures. The calculations have been carried out with analytical gradients at AM1 and PM3 semiempirical procedures, ab initio at HF level with 3-21G, 4-31G, 6-31G, and 6-31G basis sets and BP86 and BLYP as density functional methods. The results of this study suggest that the endo relative orientation on the substrate imposed by the active site is optimal in polarizing the C4-Ht bond and situating the system in the neighborhood of the quadratic region of the transition structure associated to the hydride transfer step on potential energy surface. The endo arrangement of the transition structure results in optimal frontier HOMO orbital interaction between NADH and FAD partners. The geometries of the transition structures and the corresponding transition vectors, that contain the fundamental information relating reactive fluctuation patterns, are model independent and weakly dependent on the level of theory used to determine them. A comparison between simple and complex molecular models shows that there is a minimal set of coordinates describing the essentials of hydride transfer step. The analysis of transition vector components suggests that the primary and secondary kinetic isotope effects can be strongly coupled, and this prompted the calculation of deuterium and tritium primary, secondary, and primary and secondary kinetic isotope effects. The results obtained agree well with experimental data and demonstrate this coupling.

Mechanistic interpretation of tryptophan fluorescence quenching in the time courses of glutamate dehydrogenase catalyzed reactions.

We have related the ratios of the protein fluorescence quenching and nucleotide absorbance time courses for the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate to identify the occurrence and sequential location of a previously demonstrated charge-transfer intermediate. Static studies showed the major portion of the fluorescence quenching signal to be due to radiationless singlet energy transfer from tryptophan to reduced coenzyme chromophores and that conformational changes contribute little to this signal. The ratio approach applied to the transient time courses shows correspondingly that, over most of the time range, the fluorescence quenching signal provides a quantitative measure of the sum of all posthydride transfer species. However, it also indicates the very early occurrence of a species of anomalous optical properties for the reaction catalyzed by the Clostridium symbiosum enzyme as well as that from bovine liver. Transient-state kinetic isotope effect time courses of both the fluorescence and the absorbance signals confirm that this species must be the prehydride charge-transfer complex in both enzyme reactions. Kinetic analysis of alpha-deuterio- and alpha-protio-L-glutamate reaction time courses proves the kinetic competence of the assignments. These results also demonstrate that the intramolecular transfer of a proton from the alpha-amino group of the substrate to an immediately adjacent aspartate carboxylate group on the enzyme is an obligatory initial event in the reactions catalyzed by both enzyme species, even though the occurrence of protein release from a critical lysine residue to the solvent occurs at different phases in those two reactions. The abnormally low intrinsic KIE required to simulate both the alpha-deuterio-L-glutamate reaction and its protio counterpart implies that the transition state of the hydride transfer step must be highly asymmetric.

Molecular basis for nonadditive mutational effects in Escherichia coli dihydrofolate reductase.

Recently, two sets of single, double, and quadruple residue changes within the hydrophobic substrate binding pocket of Escherichia coli dihydrofolate reductase (5,6,7,8-tetrahydrofolate+ oxidoreductase, EC 1.5.1.3) were shown to exhibit nonadditive mutational effects [Huang, Z., Wagner, C. R., & Benkovic, S. J. (1994) Biochemistry 33, 11576--11585]. In particular, the analysis of data for the L28Y, L54F, and L28Y-L54F mutations revealed nonadditive changes in the free energy associated with the substrate and cofactor binding, hydride transfer, and product release steps. Construction of a related set of mutant proteins including L28F and L28F-L54F permits a comparison of similar energy changes and provides a means for assessing differences in the interactions of Phe28 and Tyr28 with both the ligands and the side chains at residue 54. We find a single functional group change, from Phe C4-H to Tyr C4-OH, can influence the additivity of mutational effects and serve as a probe to monitor the appearance of differing enzyme conformations along the reaction pathway through changes in the interaction energy (delta GI). The comparison of additivity/nonadditivity in free energy changes for three interrelated double mutational cycles (WT --> L28F-L54F, WT --> L28Y-L54F, and L28F --> l28Y-L54F) demonstrates that the side chains of positions 28 and 54 interact cooperatively to facilitate hydride transfer by preferentially influencing the enzyme--substrate ground-state complexes. The delta GI data for individual steps also provide evidence for multiple conformations of the enzyme operating during the catalytic cycle. The fact that there are no published examples of the synergistic enhancement of favorable mutational effects is consistent with the expectation that the binding/active site surface of wild-type dihydrofolate reductase has been optimized.

Human aldose reductase: rate constants for a mechanism including interconversion of ternary complexes by recombinant wild-type enzyme.

We have used transient kinetic data for partial reactions of recombinant human aldose reductase and simulations of progress curves for D-xylose reduction with NADPH and for xylitol oxidation with NADP+ to estimate rate constants for the following mechanism at pH 8.0: E<-->E.NADPH<-->*E.NADPH<-->*E.NADPH.RCHO<-->*E.NADP+.RCH2OH <-->*E.NADP+<--> E.NADP+<-->E. The mechanism includes kinetically significant conformational changes of the two binary E.nucleotide complexes which correspond to the movement of a crystallographically identified nucleotide-clamping loop involved in nucleotide exchange. The magnitude of this conformational clamping is substantial and results in a 100- and 650-fold lowering of the nucleotide dissociation constant in the productive *E.NADPH and *E.NADP+ complexes, respectively. The transient reduction of D-xylose displays burst kinetics consistent with the conformational change preceding NADP+ release (*E.NADP+-->E.NADP+) as the rate-limiting step in the forward direction. The maximum burst rate also displays a large deuterium isotope effect (Dkburst = 3.6-4.1), indicating that hydride transfer contributes significantly to rate limitation of the sequence of steps up to and including release of xylitol. In the reverse reaction, no burst of NADPH production is observed because the hydride transfer step is overall 85% rate-limiting. Even so, the conformational change preceding NADPH release (*E.NADPH-->E.NADPH) still contributes 15% to the rate limitation for reaction in this direction. The estimated rate constant for hydride transfer from NADPH to the aldehyde of D-xylose (130 s-1) is only 5- to 10-fold lower than the corresponding rate constant determined for NADH-dependent carbonyl reduction catalyzed by lactate or liver alcohol dehydrogenase. Hydride transfer from alcohol to NADP+ (0.6 s-1), however, is at least 100- to 1000-fold slower than NAD(+)-dependent alcohol oxidation mediated by these two enzymes, resulting in a bound-state equilibrium constant for aldose reductase which greatly favors the forward reaction. The proposed kinetic model provides a basic set of rate constants for interpretation of kinetic results obtained with aldose reductase mutants generated for the purpose of examining structure-function relationships of different components of the native enzyme.

Analysis of the kinetic mechanism of enterococcal NADH peroxidase reveals catalytic roles for NADH complexes with both oxidized and two-electron-reduced enzyme forms.

Anaerobic titrations of the two-electron-reduced NADH peroxidase (EH2) with NADH and 3-acetylpyridine adenine dinucleotide (AcPyADH) yield the respective complexes without significant formation of the four-electron-reduced enzyme (EH4). Further analysis of the EH2/EH4 redox couple, however, yields a midpoint potential of -312 mV for the free enzyme at pH 7. The catalytic mechanism of the peroxidase has been evaluated with a combination of kinetic and spectroscopic approaches, including initial velocity and enzyme-monitored turnover measurements, anaerobic stopped-flow studies of the reactions of both oxidized enzyme (E) and EH2 with NADH and AcPyADH, and diode-array spectral analyses of both the reduction of E-->EH2 by NADH and the formation of EH2.NADH. Overall, these results are consistent with rapid formation of an E.NADH complex with distinct spectral properties and a rate-limiting hydride transfer step that yields EH2, with no direct evidence for intermediate FADH2 formation. The EH2.NADH complex described previously [Poole, L. B., & Claiborne, A. (1986) J. Biol. Chem. 261, 14525-14533] is not catalytically competent and reacts relatively slowly with H2O2. Stopped-flow analyses do, however, support the very rapid formation of an EH2.NADH* intermediate, with spectral properties that distinguish it from the static EH2.NADH form, and yield a first-order rate constant for the conversion between the two species that is smaller than kcat. The combined rapid-reaction and steady-state data are best accommodated by a limiting type of ternary complex mechanism very similar to that proposed previously [Parsonage, D., Miller, H., Ross, R.P., & Claiborne, A. (1993) J. Biol. Chem. 268, 3161-3167].

Metal ion activator effects on intrinsic isotope effects for hydride transfer from decarboxylation in the reaction catalyzed by the NAD-malic enzyme from Ascaris suum.

The mechanism of the oxidative decarboxylation reaction catalyzed by the NAD-malic enzyme from Ascaris suum has been examined with several different divalent metal ion activators and dinucleotide substrates. Primary deuterium and tritium isotope effects have been obtained and, in combination with the partitioning ratios of the oxalacetate intermediate to malate and pyruvate, have been used to calculate commitment factors, intrinsic deuterium isotope effects on the hydride transfer step, and intrinsic 13C isotope effects for the decarboxylation step. A survey of malate analogs has been undertaken to define the geometry of the active site and to identify functional groups on malate important for substrate binding. With NAD as dinucleotide substrate, a direct correlation between the size of the divalent metal ion activator and the intrinsic deuterium isotope effect is observed. An isotope effect significantly greater than the semiclassical limit is seen when Cd2+ is the metal ion activator, indicating a substantial tunneling contribution. The primary intrinsic 13C isotope effect on the decarboxylation step increases over the series Mg2+ < Mn2+ < Cd2+, which is in contrast to the equal isotope effects measured for these metal ions for the nonenzymatic decarboxylation of oxalacetate [Grissom, C. B., & Cleland, W. W. (1986) J. Am. Chem. Soc. 108, 5582]. With Mn2+ or Cd2+ as the divalent metal ion activator, the data support a stepwise mechanism for the enzymatic oxidative decarboxylation with NAD as the dinucleotide substrate, but a change to a concerted mechanism is indicated with more redox-positive dinucleotide substrates as suggested previously with Mg2+ as activator [Karsten, W. E., & Cook, P. F. (1994) Biochemistry 33, 2096].(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of Glutamate 268 in the Active Site of Human Liver Mitochondrial (Class 2) Aldehyde Dehydrogenase As Probed by Site-Directed Mutagenesis

On the basis of chemical modification studies, it was postulated that glutamate 268 was a component of the active site of liver aldehyde dehydrogenase [Abriola, D. P., Fields, R., MacKerell, A. D., Jr., & Pietruszko, R. (1987) Biochemistry 26, 5679-5684]. To study its role, the residue in human liver mitochondrial (class 2) aldehyde dehydrogenase was mutated to an aspartate, a glutamine, or a lysine, and the enzyme was expressed in Escherichia coli. The mutations did not affect the Km values for NAD or propionaldehyde, but grossly affected the catalytic activity of the enzymes when compared to recombinantly expressed native enzyme; the mutant enzymes had less that 0.4% of the specific activity of the recombinantly expressed native aldehyde dehydrogenase. The mutations also caused a long lag phase to occur prior to the steady state phase of the reaction. The activity of the mutant enzymes could not be restored by the addition of general bases such as sodium acetate, sodium formate, or imidazole. The Kd for NADH was essentially identical for the E268Q mutant and native enzyme. The three mutant forms of the enzyme possessed less than 0.8% of the esterolytic activity of the recombinantly expressed native enzyme. Pre-steady state analysis showed that there was no burst of NADH formation in the dehydrogenase reaction or of p-nitrophenol formation in the esterase reaction. This can be interpreted as implying that glutamate 268 may function as a general base necessary for the initial activation of the essential cysteine residue (302), rather than being involved in only the deacylation or hydride transfer step.(ABSTRACT TRUNCATED AT 250 WORDS)

Am1 and pm3 transition structure for the hydride transfer. A model of reaction catalyzed by dihydrofolate reductase

The transition state structure for the hydride transfer in dihydrofolate reductase, DHFR, enzyme has been calculated with analytical gradients at semiempirical levels: AM1 and PM3. The geometry, electronic structure and transition vector components are qualitatively semiempirical level independent. Comparing the transition structures for the hydride transfer step in models of liver alcohol dehydrogenase, formate dehydrogenase, lactate dehydrogenase, and glutathione reductase, the geometries of these stationary points are transferable and invariant. The topology of the transition structures in these enzymes resembles the one calculated in this paper.

The demonstration of a glutamate dehydrogenase-NADP-L-glutamate charge-transfer complex and its location on the reaction pathway.

In previous transient state kinetic work from this laboratory, we proposed a new mechanism for the glutamate dehydrogenase-catalyzed oxidative deamination reaction involving an initial replacement of a proton from lysine 126 by a single bound water molecule, followed by closure of the active site cleft and expulsion of bulk water, providing a hydrophobic environment for the ensuing hydride transfer step. Here, we report the results of further transient state fluorescence, absorbance, and kinetic isotope effect studies, which demonstrate the occurrence of an unusual intermediate in the early steps of that reaction. This phenomenon is revealed by an initial fluorescence burst that occurs in the time period where the absorbance signal is still in its lag phase. Using an extension of the proton/product ratio approach we have described earlier, we show that this intermediate is a strongly fluorescent but weakly absorbing species whose absorption maximum is red-shifted beyond that of other known complexes of this enzyme. The transient state kinetic isotope effects of the fluorescence and absorbance signals are compatible only with a reaction scheme in which the formation of the fluorescent complex precedes the hydride transfer step. The optical properties of this enzyme-oxidized coenzyme-substrate intermediate strongly suggest that it is a charge-transfer complex, similar in nature to the complex responsible for the well known "Racker band" reported in 1952 for glyceraldehyde-3-phosphatase dehydrogenase (Racker, E., and Krimsky, I. (1952) Nature 169, 1043-1044). The crystal structure studies of the enzyme-coenzyme and enzyme-L-glutamate complexes of the closely analogous Clostridium symbosium glutamate dehydrogenase, reported by the Sheffield group (Stillman, T. J., Baker, P. J., Britton, K. L., and Rice, D. W. (1993) J. Mol. Biol. 234, 1131-1139), provide a basis for a physical explanation of the phenomenon. We conclude that the charge transfer phenomenon is caused by the near apposition of the unprotonated alpha-amino group of the substrate in a form of the enzyme in which a conformational change has caused the complete closing of the active site cleft.

Solvent isotope effects and the nature of electrophilic catalysis m the action of the lactate dehydrogenase of bacillus stearothermophilus

Deuterium oxide at atom fractions of deuterium from 0.0 to 0.97 has an effect of less than 20% on the kinetic term k cat/ K mB (believed to reflect the transition state for the hydride-transfer step) for the reduction of pyruvic acid by NADH at 55 °C, with catalysis by the tetrameric form of the lactate dehydrogenase of Bacillus stearothermophilus. This observation suggests that the hydride-transfer event is not assisted by protonic bridging to the carbonyl group being reduced. The results are consistent with protonic bridging only if an opposing isotope effect is present, for example from a generalized conformation or solvation change. The results are consistent with other forms of electrophilic catalysis.

Computational educidation of the catalytic mechanism for ketone reduction by an oxazaborolidine–borane adduct

The reaction pathway around the complete catalytic cycle for THF–borane reduction of propanone catalysed by the oxazaborolidine form (S)-proline has been determined by AM1 semiepirical MO calculations, including characterisation of all transition structures; the transition structure for ketone coordination is almost as high in energy as that for the rate-limiting hydride-transfer step, and the calculated kinetic isotope effect for reduction by THF–[2H3]borane is in accord with experiment.

Theoretical kinetic isotope effects for the hydride-transfer step in lactate dehydrogenase

The transition-state (TS) structure for the hydride transfer in lactate dehydrogenase (LDH) enzyme has been calculated with analytical gradients at MNDO, AM1 and PM3 semiempirical levels. The TS is a first-order saddle point on the hypersurface. The geometrical arrangement of the TS results in optimal frontier orbital interaction. Information extracted from these structures is used to calculate theoretical kinetic isotope effects (KIE). The results are independent of the semiempirical procedure. The transition vector suggests that primary and secondary KIEs must be strongly coupled. The relative orientation imposed by the active-site constraints on the reactants in LDH (endo configuration) is optimal for the hydride transfer and situates the system in the neighbourhood of the saddle point.

Bovine lens aldose reductase. pH-dependence of steady-state kinetic parameters and nucleotide binding.

The pH-dependence of nucleotide binding and steady-state kinetic parameters of aldehyde reduction and alcohol oxidation catalyzed by bovine lens aldose reductase were studied. The maximal velocity of aldehyde reduction with NADPH and p-chlorobenzaldehyde was pH independent at low pH but decreased at high pH with a pK of 7.6. The V/K of NADPH displayed a bell-shaped dependence on pH and decreased with a pKa of 5.3 and a pKb of 7.5. The dissociation constant of NADPH and 3-acetylpyridine adenine dinucleotide phosphate (3-APADP) increased at low pH with a pK of 5.6-5.8 and at high pH with a pK of 9.4-9.7. The pKi of NADP and NADPH decreased below a pH of 5 and 6.7 and above a pH of 8.5 and 9.7, respectively. The pK of 8.5-9.7 appears to be due to the interaction of the 2'-phosphate of the nucleotide with a protonated base, possibly a lysine residue. The maximal velocity of alcohol oxidation was pH independent at high pH but decreased at low pH with a pK of 6.5-7.0, when p-chlorobenzyl alcohol or benzyl alcohol and 3-APADP were used. The amino acid residue for alcohol binding has a pK of 7.5-8.2 and also appears in pKi profiles of sorbinil, a competitive inhibitor versus the alcohol. Large (3-3.5) isotope effects on maximal velocity obtained with benzyl alcohol and 3-APADP suggest that with these substrates the hydride transfer step is rate-limiting and a pK of 6.5-7.0 may be the true pK of the acid-base catalyst, possibly a histidine.

Isomerization of Alkanes on Sulfated Zirconia: Promotion by Pt and by Adamantyl Hydride Transfer Species

Sulfated zirconia is a strong solid acid that catalyzes isomerization of alkanes when metal crystallites are also present. Isomerization of C 7 and higher alkanes leads to high cracking selectivities on Pt/ZrO 2-SO 4 and other solid and liquid acids, limiting industrial isomerization practice to C 4-C 6 feeds. For example, n-hexane isomerizes to isohexanes at 473 K with 99% selectivity on Pt/ZrO 2-SO 4 but n-heptane isomerization selectivities are only about 50% even at low conversions (<20%). Our work shows that hydride transfer species, such as adamantane, increase isomerization rates and inhibit CC scission reactions. n-Heptane isomerization rates show positive hydrogen kinetic orders, suggesting that the reaction proceeds on Pt/ZrO 2-SO 4 via chain transfer pathways, in which carbenium ions propagate, after a chain initiation step involving loss of hydrogen from alkanes, by hydride transfer from neutral species to carbocations. These pathways contrast with those involved in the bifunctional (metal-acid) catalytic sequences usually required for alkane isomerization, in which metal sites catalyze alkane dehydrogenation and acid sites catalyze skeletal rearrangements of alkenes. Rate-limiting hydride transfer steps are consistent with the strong influence of molecular hydride transfer agents such as adamantane, which act as co-catalysts and increase isomerization rate and selectivity. The addition of small amounts of adamantane (0.1-0.8 wt%) to n-heptane increases isomerization rates by a factor of 3 and inhibits undesirable cracking reactions. Adamantane increases hydride transfer and carbenium ion termination rates, thus reducing the surface residence time required for a catalytic turnover. As a result, desorption occurs before secondary cracking of isomerized carbenium ions. Less effective hydride transfer agents ( n-alkanes, isoalkanes) also increase n-alkane isomerization rate and selectivity, but require much higher concentrations than adamantane. Dihydrogen also acts as a hydride source in alkane isomerization catalysis, but it requires the additional presence of metals or reducible oxides, which catalyze H 2 dissociation and the formation of hydridic and protonic forms of hydrogen.

Molecular orbital studies of enzyme mechanisms. II. Catalytic oxidation of alcohols by liver alcohol dehydrogenase

AbstractSemiempirical (AM1) molecular orbital theory has been used to investigate the oxidation of alcohols at the active site of liver alcohol dehydrogenase (LADH). The model active site consists of a zinc dication coordinated to two methyl‐mercaptans (Cys‐46, Cys‐176), an imidazole (His‐67), and a water. An imidazole (His‐51) hydrogen bonded to a hydroxy‐acetate (Ser‐48) forms the remote base. AM1 calculations that address the two distinct steps in the catalytic mechanism of ethanol oxidation by LADH are reported. These two steps are: (1) the deprotonation of ethanol by imidazole (His‐51) via hydrogen‐bonded hydroxy‐acetate (Ser‐48), creating a proton relay system; and (2) the rate‐limiting hydride transfer step from ethanol C1 to nicotinamide adenine dinucleotide (NAD+), leading to product formation. Detailed calculations have been used to resolve the unsolved problems of mechanisms that have been suggested on the basis of kinetic data and crystal structures of several LADH complexes. We investigated two possible mechanisms for the deprotonation of ethanol, by zinc‐bound OH− and by direct deprotonation of zinc‐bound ethanol by imidazole via hydroxyacetate (Ser‐48). Our calculations show that there is no need for LADH to activate a water molecule at the active site as in many other zinc enzymes. This result agrees with experimental evidence. Our calculations also indicate that substrates are bound in an inner‐sphere‐pentacoordinated complex to the active site zincion. In this case, spectroscopic investigations agree with our results but crystallographic data do not. The highest activation energy is found for the hydride transfer, in agreement with the experiment. Finally, we proposed an alternative mechanism for the mode of action of LADH based upon our results. © 1993 John Wiley &amp; Sons, Inc.

Carboxymethylation-induced activation of bovine lens aldose reductase.

Carboxymethylation of bovine lens aldose reductase with 10 mM iodoacetate for 1 h at 25 degrees C led to a more than 4-fold increase in kcat. Carboxymethylation led to a 3- to 5-fold increase in Km NADPH and Km D-glyceraldehyde, whereas Km L-glyceraldehyde increased approx. 30-fold. Activation of the enzyme on carboxymethylation was accompanied by a decrease in the sensitivity of the enzyme to inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), sorbinil (Kii increased from 0.4 to 109 microM) and NADP (Kis increased from 0.01 to 0.03 mM), but not tolrestat. Activation of the enzyme was almost completely prevented by NADPH and to a lesser extent by DL-glyceraldehyde. Carboxymethylation of the enzyme did not result in the generation of several partially oxidized enzyme species, indicating the absence of partially carboxymethylated forms. Primary deuterium isotope effects on the reduced enzyme were consistent with a preferred ordered kinetic reaction scheme, in which hydride transfer is not rate limiting. The hydride transfer step does not seem to be significantly affected by carboxymethylation, nor do changes in the substrate binding steps seem to contribute to the observed rate enhancement. Increase in the turnover number of the enzyme on carboxymethylation appears to be due to facilitation of the isomerization of the E:NADP binary complex. The differential effect of carboxymethylation on sorbinil and tolrestat suggests distinct inhibitor sites on the enzyme, an S-site that binds sorbinil and a T-site that binds tolrestat.

A slow obligatory proton release step precedes hydride transfer in the liver glutamate dehydrogenase catalytic mechanism

We have used the stopped-flow indicator dye method to measure proton release and product formation simultaneously in the initial transient-state portion of the glutamate dehydrogenase-catalyzed oxidative deamination of L-glutamate. We observe a measurably slow release of a proton from the enzyme-NADP-L-glutamate complex. This proton release precedes the hydride transfer step, as indicated by the distinct lag in the product formation signal. We show that the proton release step corresponds to an obligatory intermediate in the reaction sequence. We also find that compounds which are competitive inhibitors of L-glutamate are capable of inducing this phenomenon. We prove that this unanticipated prehydride transfer event cannot be due to the release of an alpha-amino group proton from the substrate.