Tightly-bound ubiquinone in the Escherichia coli respiratory Complex I

NADH:ubiquinone oxidoreductase (Complex I), the electron input enzyme in the respiratory chain of mitochondria and many bacteria, couples electron transport to proton translocation across the membrane. Complex I is a primary proton pump; although its proton translocation mechanism is yet to be known, it is considered radically different from any other mechanism known for redox-driven proton pumps: no redox centers have been found in its membrane domain where the proton translocation takes place. Here we studied the properties and the catalytic role of the enzyme-bound ubiquinone in the solubilized, purified Complex I from Escherichia coli. The ubiquinone content in the enzyme preparations was 1.3±0.1 per bound FMN residue. Rapid mixing of Complex I with NADH, traced optically, demonstrated that both reduction and re-oxidation kinetics of ubiquinone coincide with the respective kinetics of the majority of Fe-S clusters, indicating kinetic competence of the detected ubiquinone. Optical spectroelectrochemical redox titration of Complex I followed at 270-280nm, where the redox changes of ubiquinone contribute, did not reveal any transition within the redox potential range typical for the membrane pool, or loosely bound ubiquinone (ca. +50-+100mV vs. NHE, pH 6.8). The transition is likely to take place at much lower potentials (E(m) ≤-200mV). Such perturbed redox properties of ubiquinone indicate that it is tightly bound to the enzyme's hydrophobic core. The possibility of two ubiquinone-binding sites in Complex I is discussed.     

Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247 +/- 0.0008 at 25 degrees C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25 degrees C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 degrees C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 degrees C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of Vmax/km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).     

Identification of the catalytic mechanism and estimation of kinetic parameters for fumarase

The enzyme fumarase catalyzes the reversible hydration of fumarate to malate. The reaction catalyzed by fumarase is critical for cellular energetics as a part of the tricarboxylic acid cycle, which produces reducing equivalents to drive oxidative ATP synthesis. A catalytic mechanism for the fumarase reaction that can account for the kinetic behavior of the enzyme observed in both isotope exchange studies and initial velocity studies has not yet been identified. In the present study, we develop an 11-state kinetic model of the enzyme based on the current consensus on its catalytic mechanism and design a series of experiments to estimate the model parameters and identify the major flux routes through the mechanism. The 11-state mechanism accounts for competitive binding of inhibitors and activation by different anions, including phosphate and fumarate. The model is identified from experimental time courses of the hydration of fumarate to malate obtained over a wide range of buffer and substrate concentrations. Further, the 11-state model is found to effectively reduce to a five-state model by lumping certain successive steps together to yield a mathematically less complex representation that is able to match the data. Analysis suggests the primary reaction route of the catalytic mechanism, with fumarate binding to the free unprotonated enzyme and a proton addition prior to malate release in the fumarate hydration reaction. In the reverse direction (malate dehydration), malate binds the protonated form of the enzyme, and a proton is generated before fumarate is released from the active site.     

Detailed enzyme kinetics in terms of biochemical species: study of citrate synthase

The compulsory-ordered ternary catalytic mechanism for two-substrate two-product enzymes is analyzed to account for binding of inhibitors to each of the four enzyme states and to maintain the relationship between the kinetic constants and the reaction equilibrium constant. The developed quasi-steady flux expression is applied to the analysis of data from citrate synthase to determine and parameterize a kinetic scheme in terms of biochemical species, in which the effects of pH, ionic strength, and cation binding to biochemical species are explicitly accounted for in the analysis of the data. This analysis provides a mechanistic model that is consistent with the data that have been used support competing hypotheses regarding the catalytic mechanism of this enzyme.     

Mitochondrial Complex I: structure, function, and implications in neurodegeneration

Mitochondrial Complex I (NADH Coenzyme Q oxidoreductase) is the least understood of respiratory complexes. In this review we emphasize some novel findings on this enzyme that are of relevance to the pathogenesis of neurodegenerative diseases. Besides Coenzyme Q (CoQ), also oxygen may be an electron acceptor from the enzyme, with generation of superoxide radical in the mitochondrial matrix. The site of superoxide generation is debated: we present evidence based on the rational use of several inhibitors that the one-electron donor to oxygen is an iron-sulphur cluster, presumably N2. On this assumption we present a novel mechanism of electron transfer to the acceptor, CoQ. Strong evidence is accumulating that electron transfer from Complex I to Complex III via CoQ is not performed by operation of the CoQ pool but by direct channelling within a super-complex including Complex I, Complex III and bound CoQ. Besides structural evidence of a Complex I -Complex III aggregate obtained by native electrophoresis, we have obtained kinetic evidence based on metabolic flux analysis, demonstrating that Complexes I and III behave as an individual enzyme. Quantitative and qualitative changes of phospholipids, including peroxidation, may affect the supercomplex formation. Complex I is deeply involved in pathological changes, including neurodegeneration. Maternally inherited mutations in mitochondrial DNA genes encoding for Complex I subunits are at the basis of Leber's Hereditary Optic Neuropathy; a decrease of electron transfer in the complex, due to the mutations, is not sufficient per se to explain the clinical phenotype, and other factors including proton translocation and oxygen radical generation have been considered of importance. Complex I changes are also involved in more common neurological diseases of the adult and old ages. In this review we discuss Parkinson's disease, where the pathogenic involvement of Complex I is better understood; the accumulated evidence on the mode of action of Complex I inhibitors and their effect on oxygen radical generation is discussed in terms of the aetiology and pathogenesis of the disease.     

pH profiles and isotope effects for aconitases from Saccharomycopsis lipolytica, beef heart, and beef liver. alpha-Methyl-cis-aconitate and threo-Ds-alpha-methylisocitrate as substrates

alpha-Methyl-cis-aconitate (cis-2-butene-1,2,3-tricarboxylate) was converted only to alpha-methylisocitrate (3-hydroxybutane-1,2,3-tricarboxylate) by aconitases from beef liver or S. lipolytica. While the kinetic parameters of beef liver (cytoplasmic) or heart (mitochondrial) aconitases did not vary over the pH range 4.9-9 with the natural substrates, and only slightly with the alpha-methyl substrates, the yeast aconitase exhibited a bell-shaped pH profile with all substrates and for binding of the competitive inhibitor, tricarballylate, with pK values around 7 and 9. The third pK of the substrates does not affect V/K, showing that these pK's are for catalytic groups on the enzyme. One of these catalytic groups presumably removes a proton to give the carbanion intermediate in the reaction, and the other protonates the hydroxyl group when it is eliminated to give water, possibly with the assistance of the Fe-S center. Beef liver aconitase showed a primary deuterium isotope effect of 1.12 (measured by equilibrium perturbation with deuterated alpha-methylisocitrate) which was pH independent and only slightly greater than the equilibrium isotope effect. Isotope effects with the yeast enzyme were also pH independent but about 1.22 on V/K (or when measured by equilibrium perturbation) and 1.7 on V. These data suggest a kinetic mechanism for beef aconitases in which product release occurs only by displacement by the substrate in a step independent of pH or of the protonation state of the substrate. With the yeast enzyme, product displacement either depends on the protonation state of the catalytic groups on the enzyme or can occur spontaneously at a finite rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of action of Drosophila melanogaster alcohol dehydrogenase.

Reported kinetic pH dependence data for alcohol dehydrogenase from Drosophila melanogaster are analyzed with regard to differences in rate behaviour between this non-metallo enzyme and the zinc-containing liver alcohol dehydrogenase present in vertebrates. For the Drosophila enzyme a mechanism of action is proposed according to which catalytic proton release to solution during alcohol oxidation occurs at the binary-complex level as an obligatory step preceding substrate binding. Such proton release involves an ionizing group with a pKa of about 7.6 in the enzyme.NAD+ complex, tentatively identified as a tyrosyl residue. The ionized form of this group is proposed to participate in the binding of alcohol substrates and to act as a nucleophilic catalyst of the subsequent step of hydride ion transfer from the bound alcohol to NAD+. Herein lie fundamental mechanistic differences between the metallo and non-metallo short chain alcohol dehydrogenases.     

A single amino acid residue controls ROS production in the respiratory Complex I from Escherichia coli

Reactive oxygen species (ROS) production by respiratory Complex I from Escherichia coli was studied in bacterial membrane fragments and in the isolated and purified enzyme, either solubilized or incorporated in proteoliposomes. We found that the replacement of a single amino acid residue in close proximity to the nicotinamide adenine dinucleotide (NADH)‐binding catalytic site (E95 in the NuoF subunit) dramatically increases the reactivity of Complex I towards dioxygen (O₂). In the E95Q variant short‐chain ubiquinones exhibit strong artificial one‐electron reduction at the catalytic site, also leading to a stronger increase in ROS production. Two mechanisms can contribute to the observed kinetic effects: (a) a change in the reactivity of flavin mononucleotide (FMN) towards dioxygen at the catalytic site, and (b) a change in the population of the ROS‐generating state. We propose the existence of two (closed and open) states of the NAD⁺‐bound enzyme as one feature of the substrate‐binding site of Complex I. The analysis of the kinetic model of ROS production allowed us to propose that the population of Complex I with reduced FMN is always low in the wild‐type enzyme even at low ambient redox potentials, minimizing the rate of reaction with O₂ in contrast to E95Q variant.     

Mechanism of N-methylation by the tRNA m1G37 methyltransferase Trm5

Trm5 is a eukaryal and archaeal tRNA methyltransferase that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to the N(1) position of G37 directly 3' to the anticodon. While the biological role of m(1)G37 in enhancing translational fidelity is well established, the catalytic mechanism of Trm5 has remained obscure. To address the mechanism of Trm5 and more broadly the mechanism of N-methylation to nucleobases, we examined the pH-activity profile of an archaeal Trm5 enzyme, and performed structure-guided mutational analysis. The data reveal a marked dependence of enzyme-catalyzed methyl transfer on hydrogen ion equilibria: the single-turnover rate constant for methylation increases by one order of magnitude from pH 6.0 to reach a plateau at pH 7.0. This suggests a mechanism involving proton transfer from G37 as the key element in catalysis. Consideration of the kinetic data in light of the Trm5-tRNA-AdoMet ternary cocrystal structure, determined in a precatalytic conformation, suggests that proton transfer is associated with an induced fit rearrangement of the complex that precedes formation of the reactive configuration in the active site. Key roles for the conserved R145 side chain in stabilizing a proposed oxyanion at G37-O(6), and for E185 as a general base to accept the proton from G37-N(1), are suggested based on the mutational analysis.     

Determination of the Catalytic Mechanism for Mitochondrial Malate Dehydrogenase

The kinetics of malate dehydrogenase (MDH) catalyzed oxidation/reduction of L-malate/oxaloacetate is pH-dependent due to the proton generated/taken up during the reaction. Previous kinetic studies on the mitochondrial MDH did not yield a consensus kinetic model that explains both substrate and pH dependency of the initial velocity. In this study, we propose, to our knowledge, a new kinetic mechanism to explain kinetic data acquired over a range of pH and substrate concentrations. Progress curves in the forward and reverse reaction directions were obtained under a variety of reactant concentrations to identify associated kinetic parameters. Experiments were conducted at physiologically relevant ionic strength of 0.17 M, pH ranging between 6.5 and 9.0, and at 25°C. The developed model was built on the prior observation of proton uptake upon binding of NADH to MDH, and that the MDH-catalyzed oxidation of NADH may follow an ordered bi-bi mechanism with NADH/NAD binding to the enzyme first, followed by the binding of oxaloacetate/L-malate. This basic mechanism was expanded to account for additional ionic states to explain the pH dependency of the kinetic behavior, resulting in what we believe to be the first kinetic model explaining both substrate and pH dependency of the reaction velocity.     

Kinetic and chemical mechanisms of shikimate dehydrogenase from Mycobacterium tuberculosis

Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD) catalyzes the fourth reaction in the shikimate pathway, the NADPH-dependent reduction of 3-dehydroshikimate. To gather information on the kinetic mechanism, initial velocity patterns, product inhibition, and primary deuterium kinetic isotope effect studies were performed and the results suggested a steady-state ordered bi-bi kinetic mechanism. The magnitudes of both primary and solvent kinetic isotope effects indicated that the hydride transferred from NADPH and protons transferred from the solvent in the catalytic cycle are not significantly rate limiting in the overall reaction. Proton inventory analysis indicates that one proton gives rise to solvent isotope effects. Multiple isotope effect studies indicate that both hydride and proton transfers are concerted. The pH profiles revealed that acid/base chemistry takes place in catalysis and substrate binding. The MtbSD 3D model was obtained in silico by homology modeling. Kinetic and chemical mechanisms for MtbSD are proposed on the basis of experimental data.     

A possible role for iron-sulfur cluster N2 in proton translocation by the NADH: ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The enzyme mechanism is still unknown due to the lack of a high-resolution structure and its complicated composition. The complex from Escherichia coli is made up of 13 subunits called NuoA through NuoN and contains one FMN and nine iron-sulfur (Fe/S) clusters as redox groups. The pH dependence of the midpoint redox potential of the Fe/S cluster named N2 and its spin-spin interaction with ubiquinone radicals made it an ideal candidate for a key component in redox-driven proton translocation. During the past years we have assigned the subunit localization of cluster N2 to subunit NuoB by site-directed mutagenesis and predicted its ligation by molecular simulation. Redox-induced FT-IR spectroscopy has shown that its redox reaction is accompanied by the protonation and deprotonation of individual amino acid residues. These residues have been identified by site-directed mutagenesis. The enzyme catalytic activity depends on the presence of cluster N2 and is coupled with major conformational changes. From these data a model for redox-induced conformation-driven proton translocation has been derived.     

Kinetic studies of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: pH variation of kinetic parameters

The specificity and kinetic parameters of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has been examined under a range of conditions in order to elucidate details about the mechanism of action of this enzyme. The rate of oxidation of glucose 6-phosphate is inhibited by the addition of various organic solvents. However, the low, inherent glucose dehydrogenase activity of this enzyme was stimulated under these conditions, and was further activated by divalent anions that were observed to be inhibitors of the glucose 6-phosphate dehydrogenation. From an examination of the pH variation of the enzyme kinetic parameters two groups on the enzyme that appear to be involved in the binding of the phosphate group of the sugar substrate have been detected. An enzyme catalytic group, probably a carboxylic acid, has been identified that accepts the proton from the hydroxyl group at carbon-1 of the sugar substrate during its oxidation to a lactone. The ionization of a group on the enzyme with a pK of 8.7 resulted in an increase in the maximum velocity of the glucose-6-phosphate dehydrogenase activity of the enzyme as a consequence of a pH-dependent product release step that is no longer rate limiting at high pH. Stabilization of gluconic acid-delta-lactone against nonenzymatic hydrolysis by organic solvents has allowed the kinetic parameters of the reverse reaction to be reliably measured for the first time in a narrow pH range.     

Substrate-dependent modulation of the enzymatic catalytic activity: Reduction of nitrate, chlorate and perchlorate by respiratory nitrate reductase from Marinobacter hydrocarbonoclasticus 617

The respiratory nitrate reductase complex (NarGHI) from Marinobacter hydrocarbonoclasticus 617 (Mh, formerly Pseudomonas nautica 617) catalyzes the reduction of nitrate to nitrite. This reaction is the first step of the denitrification pathway and is coupled to the quinone pool oxidation and proton translocation to the periplasm, which generates the proton motive force needed for ATP synthesis. The Mh NarGH water-soluble heterodimer has been purified and the kinetic and redox properties have been studied through in-solution enzyme kinetics, protein film voltammetry and spectropotentiometric redox titration. The kinetic parameters of Mh NarGH toward substrates and inhibitors are consistent with those reported for other respiratory nitrate reductases. Protein film voltammetry showed that at least two catalytically distinct forms of the enzyme, which depend on the applied potential, are responsible for substrate reduction. These two forms are affected differentially by the oxidizing substrate, as well as by pH and inhibitors. A new model for the potential dependence of the catalytic efficiency of Nars is proposed.     

Intermediate forms of cytochrome oxidase observed in transient kinetic experiments and those visited in the catalytic cycle

The cytochrome oxidase family of heme-copper oxidases has been the subject of intense kinetic and mechanistic enquiry. Much of this work has focussed on transient kinetic studies of the partial reactions of the enzyme with the goal being to build a kinetic model describing the catalytic cycle that the enzyme undergoes to direct the oxidation of substrate, reduction of oxygen and vectorial proton transfer. A key aspect of such a model is to define the structures of each of the intermediate forms the enzyme takes up as it traverses the catalytic cycle. One complication that has been prevalent with mitochondrial cytochrome c oxidase is the existence of structural variants of the enzyme, as isolated, that may not be participants in catalysis. Studies of structurally simpler procaryotic members of the family may offer new insight on the intermediates of catalysis. In this paper transient-state and steady-state kinetic studies of cytochrome aa(3)-600 from Bacillus subtilis are integrated into a model of the catalytic cycle. This model specifies that the P intermediate accumulates in the steady-state and it is proposed that the step following its formation is limited by proton uptake.     

Catalytic roles of divalent metal ions in phosphoryl transfer by EcoRV endonuclease.

The rate constant for the phosphoryl transfer step in site-specific DNA cleavage by EcoRV endonuclease has been determined as a function of pH and identity of the required divalent metal ion cofactor, for both wild-type and T93A mutant enzymes. These measurements show bell-shaped pH-rate curves for each enzyme in the presence of Mg2+ as a cofactor, indicating general base catalysis for the nucleophilic attack of hydroxide ion on the scissile phosphate, and general acid catalysis for protonation of the leaving 3'-O anion. The kinetic data support a model for phosphoryl transfer based on wild-type and T93A cocrystal structures, in which the ionizations of two distinct metal-ligated waters respectively generate the attacking hydroxide ion and the proton for donation to the leaving group. The model concurs with recent observations of two metal ions bound in the active sites of the type II restriction endonucleases BamHI and BglI, suggesting the possibility of a similar catalytic mechanism functioning in many or all members of this enzyme family.     

Sodium-translocating NADH:quinone oxidoreductase as a redox-driven ion pump

The Na⁺-translocating NADH:ubiquinone oxidoreductase (Na⁺-NQR) is a component of the respiratory chain of various bacteria. This enzyme is an analogous but not homologous counterpart of mitochondrial Complex I. Na⁺-NQR drives the same chemistry and also uses released energy to translocate ions across the membrane, but it pumps Na⁺ instead of H⁺. Most likely the mechanism of sodium pumping is quite different from that of proton pumping (for example, it could not accommodate the Grotthuss mechanism of ion movement); this is why the enzyme structure, subunits and prosthetic groups are completely special. This review summarizes modern knowledge on the structural and catalytic properties of bacterial Na⁺-translocating NADH:quinone oxidoreductases. The sequence of electron transfer through the enzyme cofactors and thermodynamic properties of those cofactors is discussed. The resolution of the intermediates of the catalytic cycle and localization of sodium-dependent steps are combined in a possible molecular mechanism of sodium transfer by the enzyme.     

The kinetic mechanisms of the bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli

The kinetic mechanisms of the reactions catalyzed by the two catalytic domains of aspartokinase-homoserine dehydrogenase I from Escherichia coli have been determined. Initial velocity, product inhibition, and dead-end inhibition studies of homoserine dehydrogenase are consistent with an ordered addition of NADPH and aspartate beta-semialdehyde followed by an ordered release of homoserine and NADP+. Aspartokinase I catalyzes the phosphorylation of a number of L-aspartic acid analogues and, moreover, can utilize MgdATP as a phosphoryl donor. Because of this broad substrate specificity, alternative substrate diagnostics was used to probe the kinetic mechanism of this enzyme. The kinetic patterns showed two sets of intersecting lines that are indicative of a random mechanism. Incorporation of these results with the data obtained from initial velocity, product inhibition, and dead-end inhibition studies at pH 8.0 are consistent with a random addition of L-aspartic acid and MgATP and an ordered release of MgADP and beta-aspartyl phosphate.     

Detailed kinetics and regulation of mammalian NAD-linked isocitrate dehydrogenase

A mathematical model is presented to describe the catalytic mechanism of mammalian NAD-linked isocitrate dehydrogenase (NAD-IDH), a highly regulated enzyme in the tricarboxylic acid cycle, a crucial pathway in energy metabolism and biosynthesis. The mechanism accounts for allosteric regulation by magnesium-bound isocitrate and EGTA and calcium-bound ATP and ADP. The developed model is used to analyze kinetic data for the cardiac enzyme and to estimate kinetic parameter values. Since the kinetic mechanism is expressed in terms of chemical species (rather than biochemical reactants), the model explicitly accounts for the effects of biochemical state (ionic strength, pH, temperature, and metal cation concentration) on the kinetics. Because the substrate isocitrate competes with allosteric activators (ATP and ADP) and an inhibitor (EGTA) for metal ion cofactors (Ca(2+) and Mg(2+)), the observed kinetic relationships between reactants, activator and inhibitor concentrations, and catalytic flux are complex. Our analysis reveals that under physiological conditions, the ADP/ATP ratio plays a more significant role than Ca(2+) concentration in regulating the enzyme's activity. In addition, the enzyme is highly sensitive to Mg(2+) concentration in the physiological range, pointing to a potential regulatory role of [Mg(2+)] in mitochondrial energy metabolism.     

Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain D-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects

Mannitol dehydrogenases (MDH) are a family of Zn(2+)-independent long-chain alcohol dehydrogenases that catalyze the regiospecific NAD(+)-dependent oxidation of a secondary alcohol group in polyol substrates. pH and primary deuterium kinetic isotope effects on kinetic parameters for reaction of recombinant MDH from Pseudomonas fluorescens with D-mannitol have been measured in H(2)O and D(2)O at 25 degrees C and used to determine the relative timing of C-H and O-H bond cleavage steps during alcohol conversion. The enzymatic rates decreased at low pH; apparent pK values for log(k(cat)/K(mannitol)) and log k(cat) were 9.2 and 7.7 in H(2)O, respectively, and both were shifted by +0.4 pH units in D(2)O. Proton inventory plots for k(cat) and k(cat)/K(mannitol) were determined at pL 10.0 using protio or deuterio alcohol and were linear at the 95% confidence level. They revealed the independence of primary deuterium isotope effects on the atom fraction of deuterium in a mixed H(2)O-D(2)O solvent and yielded single-site transition-state fractionation factors of 0.43 +/- 0.05 and 0.47 +/- 0.01 for k(cat)/K(mannitol) and k(cat), respectively. (D)(k(cat)/K(mannitol)) was constant (1.80 +/- 0.20) in the pH range 6.0-9.5 and decreased at high pH to a limiting value of approximately 1. Measurement of (D)(k(cat)/K(fructose)) at pH 10.0 and 10.5 using NADH deuterium-labeled in the 4-pro-S position gave a value of 0.83, the equilibrium isotope effect on carbonyl group reduction. A mechanism of D-mannitol oxidation by MDH is supported by the data in which the partly rate-limiting transition state of hydride transfer is stabilized by a single solvation catalytic proton bridge. The chemical reaction involves a pH-dependent internal equilibrium which takes place prior to C-H bond cleavage and in which proton transfer from the reactive OH to the enzyme catalytic base may occur. Loss of a proton from the enzyme at high pH irreversibly locks the ternary complex with either alcohol or alkoxide bound in a conformation committed of undergoing NAD(+) reduction at a rate about 2.3-fold slower than the corresponding reaction rate of the protonated complex. Transient kinetic studies for D-mannitol oxidation at pH(D) 10.0 showed that the solvent isotope effect on steady-state turnover originates from a net rate constant of NADH release that is approximately 85% rate-limiting for k(cat) and 2-fold smaller in D(2)O than in H(2)O.