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.     

Structures of the dI2dIII1 complex of proton-translocating transhydrogenase with bound, inactive analogues of NADH and NADPH reveal active site geometries

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The enzyme comprises three components; dI binds NAD(H), dIII binds NADP(H), and dII spans the membrane. The 1,4,5,6-tetrahydro analogue of NADH (designated H2NADH) bound to isolated dI from Rhodospirillum rubrum transhydrogenase with similar affinity to the physiological nucleotide. Binding of either NADH or H2NADH led to closure of the dI mobile loop. The 1,4,5,6-tetrahydro analogue of NADPH (H2NADPH) bound very tightly to isolated R. rubrum dIII, but the rate constant for dissociation was greater than that for NADPH. The replacement of NADP+ on dIII either with H2NADPH or with NADPH caused a similar set of chemical shift alterations, signifying an equivalent conformational change. Despite similar binding properties to the natural nucleotides, neither H2NADH nor H2NADPH could serve as a hydride donor in transhydrogenation reactions. Mixtures of dI and dIII form dI2dIII1 complexes. The nucleotide charge distribution of complexes loaded either with H2NADH and NADP+ or with NAD+ and H2NADPH should more closely mimic the ground states for forward and reverse hydride transfer, respectively, than previously studied dead-end species. Crystal structures of such complexes at 2.6 and 2.3 A resolution are described. A transition state for hydride transfer between dihydronicotinamide and nicotinamide derivatives determined in ab initio quantum mechanical calculations resembles the organization of nucleotides in the transhydrogenase active site in the crystal structure. Molecular dynamics simulations of the enzyme indicate that the (dihydro)nicotinamide rings remain close to a ground state for hydride transfer throughout a 1.4 ns trajectory.     

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.     

Hydride transfer stereospecificity of rat liver aldehyde dehydrogenases

The stereospecificity of hydride transfer to NAD+ by several forms of rat liver aldehyde dehydrogenase was determined by a nuclear magnetic resonance method. The forms included several mitochondrial and microsomal isozymes from normal liver, as well as isozymes from xenobiotic-treated and tumor cells. The proton added to NAD+ comes exclusively from the aldehyde substrate and in all cases was A (pro-R)-stereospecific.     

Mechanism of Salmonella typhimurium histidinol dehydrogenase: kinetic isotope effects and pH profiles.

L-Histidinol dehydrogenase catalyzes the biosynthetic oxidation of L-histidinol to L-histidine with sequential reduction of two molecules of NAD. Previous isotope exchange results had suggested that the oxidation of histidinol to the intermediate histidinaldehyde occurred 2-3-fold more rapidly than overall catalysis. In this work, we present kinetic isotope effects (KIE) studies at pH 9.0 and at pH 6.7 with stereospecifically mono- and dideuterated histidinols. The data at pH 9.0 support minimal participation of the first hydride transfer and substantial participation of the second hydride transfer in the overall rate limitation. Stopped-flow experiments with protiated histidinol revealed a small burst of NADH production with stoichiometry of 0.12 per subunit, and 0.25 per subunit with dideuterated histidinol, indicating that the overall first half-reaction was not significantly faster than the second reaction sequence. Results from kcat and kcat/KM titrations with histidinol, NAD, and the alternative substrate imidazolyl propanediol demonstrated an essential base with pKa values between 7.7 and 8.4. In KIE experiments performed at pH 6.7 or with a coenzyme analogue at pH 9. 0, the first hydride transfer became more rate limiting. Kinetic simulations based on rate constants estimated from this work fit well with a mechanism that includes a relatively fast, and thermodynamically unfavorable, hydride transfer from histidinol and a slower, irreversible second hydride transfer from a histidinaldehyde derivative. Thus, although the chemistry of the first hydride transfer is fast, both partial reactions participate in the overall rate limitation.     

Effect of pH on the liver alcohol dehydrogenase reaction.

New transient kinetic methods, which allow kinetics to be carried out under conditions of excess substrate, have been employed to investigate the kinetics of hydride transfer from NADH to aromatic aldehydes and from aromatic alcohols to NAD+ as a function of pH. The hydride transfer rate from 4-deuterio-NADH to beta-naphthaldehyde is nearly pH independent from pH 6.0 to pH 9.9; the isotope effect is also pH independent with kappa-H/kappaD congruent to 2.3. Likewise, the rate of oxidation of benzyl alcohol by NAD+ changes little with pH between pH 8.75 and pH 5.9; the isotope effect for this process is between 3.0 and 4.4. Earlier substituent effect studies on the reduction of aromatic aldehydes were consistent with electrophilic catalysis by either zinc or a protonic acid. The pH independence of hydride transfer is consistent with electrophilic catalysis by zinc since such catalysis by protonic acid (with a pK between 6.0 and 10.0) would show strong pH dependence. However, protonic acid catalysis cannot be excluded if the pKa of the acid catalyst in the ternary NADH-E-RCOH complex were smaller than 6.0 or smaller than 10.0. The two kinetic parameters changing significantly with pH are the kinetic binding constant for ternary complex formation with aromatic alcohol and the rate of dissociation of aromatic alcohols from enzyme. This is consistent with base-catalyzed removal of a proton from alcohol substrated and consequent acid catalysis of protonation of a zinc-alcoholate complex. The equilibrium constant for hydride transfer from benzaldehyde to benzyl alcohol at pH 8.75 is K-eq equals kappa-H/kappa-H equals 42; this constant has important consequences concerning subunit interactions during liver alcohol dehydrogenase catalysis.     

Mechanism of urocanase as studied by deuterium isotope effects and labeling patterns

Nicotinamide adenine dinucleotide (NAD) dependent urocanase (4'-imidazolone-5'-propionate hydro-lyase, EC 4.2.1.49) from Pseudomonas putida was found to catalyze an exchange reaction between solvent and the 4'-hydrogen of urocanate or imidazolepropionate at a rate faster than that of overall deuterium was compared to unlabeled urocanate as a substrate, no isotope rate effect was noted. For examination of the possibility of an NAD+-mediated intramolecular hydride transfer of the 4'-hydrogen to a position on the side chain of oxoimidazolepropionate, the origins of hydrogen at positions 2 and 3 in the propionate chain were studied as a function of reaction time and extent of exchange of the 4'-hydrogen. No transfer of hydrogen from the 4' position to the side chain was observed, thereby eliminating mechanisms requiring hydride transfer via NADH between these positions. Catalytic rates in 1H2O vs. 2H2O revealed a 3-fold difference which was ascribed to a rate-limiting proton addition step. Similarly, a 5-fold decrease in Vmax was found for the reverse reaction when oxoimidazole[2,3-2H2]propionate was compared to unlabeled oxoimidazolepropionate. These data support a mechanism involving water addition across the conjugated double bond system of urocanate, rather than an internal oxidation--reduction process, yet NAD+ is required. A mechanism is proposed which uses electron delocalization in the imidazole nucleus, via an imidazole--NAD adduct, to facilitate water attack and subsequent formation of oxoimidazolepropionate.     

Mechanism and applications of phosphite dehydrogenase

Phosphite dehydrogenase catalyzes the NAD+-dependent oxidation of hydrogen phosphonate (common name phosphite) to phosphate in what amounts to a formal phosphoryl transfer reaction from hydride to hydroxide. This review places the enzyme in the context of phosphorus redox metabolism in nature and discusses the results of mechanistic investigations into its reaction mechanism. The potential of the enzyme as a NAD(P)H cofactor regeneration system is discussed as well as efforts to engineer the cofactor specificity of the protein.     

Crystal structure at 1.8 A resolution of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis

CDP-D-glucose 4,6-dehydratase catalyzes the conversion of CDP-D-glucose to CDP-4-keto-6-deoxyglucose in an NAD(+)-dependent manner. The product of this conversion is a building block for a variety of primary antigenic determinants in bacteria, possibly implicated directly in reactive arthritis. Here, we describe the solution of the high-resolution crystal structure of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis in the resting state. This structure represents the first CDP nucleotide utilizing dehydratase of the short-chain dehydrogenase/reductase (SDR) family to be determined, as well as the first tetrameric structure of the subfamily of SDR enzymes in which NAD(+) undergoes a full reaction cycle. On the basis of a comparison of this structure with structures of homologous enzymes, a chemical mechanism is proposed in which Tyr157 acts as the catalytic base, initiating hydride transfer by abstraction of the proton from the sugar 4'-hydroxyl. Concomitant with the removal of the proton from the 4'-hydroxyl oxygen, the sugar 4'-hydride is transferred to the B face of the NAD(+) cofactor, forming the reduced cofactor and a CDP-4-keto-d-glucose intermediate. A conserved Lys161 most likely acts to position the NAD(+) cofactor so that hydride transfer is favorable and/or to reduce the pK(a) of Tyr157. Following substrate oxidation, we propose that Lys134, acting as a base, would abstract the 5'-hydrogen of CDP-4-keto-D-glucose, priming the intermediate for the spontaneous loss of water. Finally, the resulting Delta(5,6)-glucoseen intermediate would be reduced suprafacially by the cofactor, and reprotonation at C-5' is likely mediated by Lys134.     

Catalytic role of proton transfers in the formation of a tetrahedral adduct in a serine carboxyl peptidase

Quantum mechanical/molecular mechanical molecular dynamics and 2D free energy simulations are performed to study the formation of a tetrahedral adduct by an inhibitor N-acetyl-isoleucyl-prolyl-phenylalaninal (AcIPF) in a serine-carboxyl peptidase (kumamolisin-As) and elucidate the role of proton transfers during the nucleophilic attack by the Ser278 catalytic residue. It is shown that although the serine-carboxyl peptidases have a fold resembling that of subtilisin, the proton transfer processes during the nucleophilic attack by the Ser residue are likely to be more complex for these enzymes compared to the case in classical serine proteases. The computer simulations demonstrate that both general base and acid catalysts are required for the formation and stabilization of the tetrahedral adduct. The 2D free energy maps further demonstrate that the proton transfer from Ser278 to Glu78 (the general base catalyst) is synchronous with the nucleophilic attack, whereas the proton transfer from Asp164 (the general acid catalyst) to the inhibitor is not. The dynamics of the protons at the active site in different stages of the nucleophilic attack as well as the motions of the corresponding functional groups are also studied. It is found that the side chain of Glu78 is generally rather flexible, consistent with its possible multifunctional role during catalysis. The effects of proton shuffling from Asp82 to Glu78 and from Glu32 to Asp82 are examined, and the results indicate that such proton shuffling may not play an important role in the stabilization of the tetrahedral intermediate analogue.     

Structural analysis of glyceraldehyde 3-phosphate dehydrogenase from Escherichia coli: direct evidence of substrate binding and cofactor-induced conformational changes

The crystal structures of gyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Escherichia coli have been determined in three different enzymatic states, NAD(+)-free, NAD(+)-bound, and hemiacetal intermediate. The NAD(+)-free structure reported here has been determined from monoclinic and tetragonal crystal forms. The conformational changes in GAPDH induced by cofactor binding are limited to the residues that bind the adenine moiety of NAD(+). Glyceraldehyde 3-phosphate (GAP), the substrate of GAPDH, binds to the enzyme with its C3 phosphate in a hydrophilic pocket, called the "new P(i)" site, which is different from the originally proposed binding site for inorganic phosphate. This observed location of the C3 phosphate is consistent with the flip-flop model proposed for the enzyme mechanism [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. Via incorporation of the new P(i) site in this model, it is now proposed that the C3 phosphate of GAP initially binds at the new P(i) site and then flips to the P(s) site before hydride transfer. A superposition of NAD(+)-bound and hemiacetal intermediate structures reveals an interaction between the hydroxyl oxygen at the hemiacetal C1 of GAP and the nicotinamide ring. This finding suggests that the cofactor NAD(+) may stabilize the transition state oxyanion of the hemiacetal intermediate in support of the flip-flop model for GAP binding.     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.     

Variation of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 1. Liver alcohol dehydrogenase with benzyl alcohol and yeast aldehyde dehydrogenase with benzaldehyde

Primary intrinsic deuterium and 13C isotope effects have been determined for liver (LADH) and yeast (YADH) alcohol dehydrogenases with benzyl alcohol as substrate and for yeast aldehyde dehydrogenase (ALDH) with benzaldehyde as substrate. These values have also been determined for LADH as a function of changing nucleotide substrate. As the redox potential of the nucleotide changes from -0.320 V with NAD to -0.258 V with acetylpyridine-NAD, the product of primary and secondary deuterium isotope effects rises from 4 toward 6.5, while the primary 13C isotope effect drops from 1.025 to 1.012, suggesting a trend from a late transition state with NAD to one that is more symmetrical. The values of Dk (again the product of primary and secondary isotope effects) and 13k for YADH with NAD are 7 and 1.023, suggesting for this very slow reaction a more stretched, and thus symmetrical, transition state. With ALDH and NAD, the primary 13C isotope effect on the hydride transfer step lies in the range 1.3-1.6%, and the alpha-secondary deuterium isotope effect on the same step is at least 1.22, but 13C isotope effects on formation of the thiohemiacetal intermediate and on the addition of water to the thio ester intermediate are less than 1%. On the basis of the relatively large 13C isotope effects, we conclude that carbon motion is involved in the hydride transfer steps of dehydrogenase reactions.     

Mouse intracellular immunoglobulin M. Structure and identification of a free thiol group

1. The formation of the non-enzymic adduct of NAD(+) and sulphite was investigated. In agreement with others we conclude that the dianion of sulphite adds to NAD(+). 2. The formation of ternary complexes of either lactate dehydrogenase or malate dehydrogenase with NAD(+) and sulphite was investigated. The u.v. spectrum of the NAD-sulphite adduct was the same whether free or enzyme-bound at either pH6 or pH8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. 3. The effect of pH on the concentration of NAD-sulphite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO(3) (-)=SO(3) (2-)+H(+)), the enzyme-bound adduct showed a bell-shaped pH-stability curve, which indicated that an enzyme side chain of pK=6.2 must be protonated for the complex to form. Since the adduct does not bind to the enzyme when histidine-195 of lactate dehydrogenase is ethoxycarbonylated we conclude that the protein group involved is histidine-195. 4. The pH-dependence of the formation of a ternary complex of lactate dehydrogenase, NAD(+) and oxalate suggested that an enzyme group is protonated when this complex forms. 5. The rate at which NAD(+) binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD(+) by rapid reaction with sulphite. The rate of NAD(+) dissociation from the enzymes was calculated from the bimolecular association kinetic constant and from the equilibrium binding constant and was in both cases much faster than the forward V(max.). No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD(+).     

Multiple conformations of NAD and NADH when bound to human cytosolic and mitochondrial aldehyde dehydrogenase

Crystallographic analysis revealed that the nicotinamide ring of NAD can bind with multiconformations to aldehyde dehydrogenase (ALDH) (Ni, L., Zhou, J., Hurley, T. D., and Weiner, H. (1999) Protein Sci. 8, 2784-2790). Electron densities can be defined for two conformations, neither of which appears to be compatible with the catalytic reaction. In one conformation, it would prevent glutamate 268 from functioning as a general base needed to activate the catalytic nucleophile, cysteine 302. In the other conformation, the nicotinamide is too far from the enzyme-substrate adduct for efficient hydride transfer. In this study, NMR and fluorescence spectroscopies were used to demonstrate that NAD and NADH bind to human liver cytosol and mitochondrial ALDH such that the nicotinamide samples a population of conformations while the adenosine region remains relatively immobile. Although the nicotinamide possesses extensive conformational heterogeneity, the catalyzed reaction leads to the stereospecific transfer of hydride to the coenzyme. Mobility allows the nicotinamide to move into position to be reduced by the enzyme-substrate adduct. Although the reduced nicotinamide ring retains mobility after NADH formation, the extent of the motion is less than that of NAD. It appears that after reduction the population of favored nicotinamide conformations shifts toward those that do not interfere with the ability of the enzyme to release the reaction product. In the case of the mitochondrial, but not the cytosolic, enzyme this change in conformational preference is promoted by the presence of Mg2+ ions. Coenzyme conformational mobility appears to be beneficial to catalysis by ALDH throughout the catalytic cycle.     

Effect of pH on the process of ternary-complex interconversion in the liver-alcohol-dehydrogenase reaction.

1. Kinetic relationships referring to multiple-turnover conditions have been derived for the slowest exponential transient appearing in two-substrate enzyme reactions proceeding by an ordered ternary-complex mechanism. The validity of these and previously derived theoretical relationships for this mechanism has been tested by application to the liver alcohol dehydrogenase reaction. 2. All essential features of the transient-state kinetics of alcohol oxidation by NAD+ in the liver alcohol dehydrogenase system can be qualitatively and quantitatively explained in view of the compulsory-order mechanism in the proposed scheme. There is no kinetic evidence for any half-of-the-sites reactivity of the enzyme. A consistent set of rate constants is reported for the enzymic oxidation of benzyl alcohol at pH 8.75. 3. Transient-state rate parameters for benzyl alcohol/benzaldehyde catalysis by liver alcohol dehydrogenase have been determined at different pH. The interpretation of such rate parameters is critically discussed with reference to their informative value for the purpose of determination of rate constants (k and k') for the process of ternary-complex interconversion in the proposed scheme. It is concluded that the apparent rate constant (k') for hydride transfer from benzyl alcohol to NAD+ is dependent on a proton dissociation step with a pKa of 6.4, whereas the rate constant (k) for hydride transfer from NADH to benzaldehyde exhibits no corresponding dependence on proton association. 4. The asymmetric pH dependence of the forward and reverse rate of ternary-complex interconversion during liver alcohol dehydrogenase catalysis appears to reflect an obligatory step of alcohol/alcoholate ion equilibration occurring at the ternary-complex level. It is suggested that the observed pKa 6.4 dependence of the transient rate of alcohol oxidation can be attributed to a coupled acid-base system involving minimally the enzyme-bound alcohol and the protein residues Ser-48 and His-51.     

Kinetic studies on the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes.

Steady-state kinetic techniques have been used to investigate each of the reactions catalyzed by the bifunctional enzyme, chorismate mutase-prephenate dehydrogenase, from Aerobacter aerogenes. The results of steady-state velocity studies in the absence of products, as well as product and dead-end inhibition studies, suggest that the prephenate dehydrogenase reaction conforms to a rapid equilibrium random mechanism which involes the formation of two dead-end complexes, viz, enzyme-NADH-prephenate and enzyme-NAD+-hydroxyphenylpyruvate. Chorismate functions as an activator of the dehydrogenase while both prephenate and hydroxyphenylpyruvate acted as competitive inhibitors in the mutase reaction. By contrast. bpth NAD+ and NADH function as activators of the mutase. Values of the kinetic parameters associated with the mutase and dehydrogenase reactions have been determined and the results discussed in terms of possible relationships between the catalytic sites for the two reactions. The data appear to be consistent with the enzyme having either a single site at which both reactions occur or two separate sites which possess similar kinetic properties.     

The role of invariant amino acid residues at the hydride transfer site of proton-translocating transhydrogenase

Transhydrogenase couples proton translocation across a membrane to hydride transfer between NADH and NADP+. Previous x-ray structures of complexes of the nucleotide-binding components of transhydrogenase ("dI2dIII1" complexes) indicate that the dihydronicotinamide ring of NADH can move from a distal position relative to the nicotinamide ring of NADP+ to a proximal position. The movement might be responsible for gating hydride transfer during proton translocation. We have mutated three invariant amino acids, Arg-127, Asp-135, and Ser-138, in the NAD(H)-binding site of Rhodospirillum rubrum transhydrogenase. In each mutant, turnover by the intact enzyme is strongly inhibited. Stopped-flow experiments using dI2dIII1 complexes show that inhibition results from a block in the steps associated with hydride transfer. Mutation of Asp-135 and Ser-138 had no effect on the binding affinity of either NAD+ or NADH, but mutation of Arg-127 led to much weaker binding of NADH and slightly weaker binding of NAD+. X-ray structures of dI2dIII1 complexes carrying the mutations showed that their effects were restricted to the locality of the bound NAD(H). The results are consistent with the suggestion that in wild-type protein movement of the Arg-127 side chain, and its hydrogen bonding to Asp-135 and Ser-138, stabilizes the dihydronicotinamide of NADH in the proximal position for hydride transfer.     

Investigation on the kinetic mechanism of octopine dehydrogenase. A regulatory behavior.

The kinetic scheme of octopine dehydrogenase of Pecten maximus L., a monomeric enzyme obeying a bi-ter sequential mechanism, was completed, essentially in the forward reaction, by steady-state studies over a wide range of substrate concentration at pH 7.0. Deviation from the Michaelis-Menten behavior with respect to NAD+ and other significant kinetic data led us to ascribe for octopine dehydrogenase mechanism the mnemonical enzyme concept. In addition, another regulatory behavior can be envisaged involving the formation of two dead-end complexes enzyme.NADH.D-octopine and enzyme.NAD+.pyruvate.L-arginine.     

Ionisations within a subtilisin-glyoxal inhibitor complex

Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a K(i)=2.3+/-0.2 microM at pH 7.0 and 25 degrees C. Using Z-Ala-Pro-[2-(13)C]Phe-glyoxal we have detected a signal at 107.3 ppm by (13)C NMR, which we assign to the tetrahedral adduct formed between the hydroxy group of serine-195 and the (13)C-enriched keto-carbon of the inhibitor. The chemical shift of this signal is pH independent from pH 4.2 to 7.0 and we conclude that the oxyanion pK(a)<3. This is the first observation of oxyanion formation in a reversible subtilisin-inhibitor complex. The inhibitor is bound as a hemiketal which is in slow exchange with the free inhibitor. Inhibitor binding depends on a pK(a) of approximately 6.5 in the free enzyme and on a pK(a)<3.0 when the inhibitor is bound to subtilisin. Protonation of the oxyanion promotes the disassociation of the inhibitor. We show that oxyanion formation cannot be rate limiting during catalysis and that subtilisin stabilises the oxyanion by at least 45.1 kJ mol(-1). We conclude that if the energy required for oxyanion stabilisation is utilised as binding energy in drug design it should make a significant contribution to inhibitor potency.