Participation of histidine-51 in catalysis by horse liver alcohol dehydrogenase

Histidine-51 in horse liver alcohol dehydrogenase (ADH) is part of a hydrogen-bonded system that appears to facilitate deprotonation of the hydroxyl group of water or alcohol ligated to the catalytic zinc. The contribution of His-51 to catalysis was studied by characterizing ADH with His-51 substituted with Gln (H51Q). The steady-state kinetic constants for ethanol oxidation and acetaldehyde reduction at pH 8 are similar for wild-type and H51Q enzymes. In contrast, the H51Q substitution significantly shifts the pH dependencies for steady-state and transient reactions and decreases by 11-fold the rate constant for the transient oxidation of ethanol at pH 8. Modest substrate deuterium isotope effects indicate that hydride transfer only partially limits the transient oxidation and turnover. Transient data show that the H51Q substitution significantly decreases the rate of isomerization of the enzyme-NAD(+) complex and becomes a limiting step for ethanol oxidation. Isomerization of the enzyme-NAD(+) complex is rate limiting for acetaldehyde reduction catalyzed by the wild-type enzyme, but release of alcohol is limiting for the H51Q enzyme. X-ray crystallography of doubly substituted His51Gln:Lys228Arg ADH complexed with NAD(+) and 2,3- or 2,4-difluorobenzyl alcohol shows that Gln-51 isosterically replaces histidine in interactions with the nicotinamide ribose of the coenzyme and that Arg-228 interacts with the adenosine monophosphate of the coenzyme without affecting the protein conformation. The difluorobenzyl alcohols bind in one conformation. His-51 participates in, but is not essential for, proton transfers in the mechanism.     

Kinetics of native and modified liver alcohol dehydrogenase with coenzyme analogues: isomerization of enzyme-nicotinamide adenine dinucleotide complex

Coenzyme analogues with the adenosine ribose replaced with n-propyl, n-butyl, and n-pentyl groups; coenzyme analogues with the adenosine replaced with 3-(4-acetylanilino)propyl and 6-(4-acetylanilino)hexyl moieties; and nicotinamide mononucleotide, nicotinamide hypoxanthine dinucleotide, and 3-acetylpyridine adenine dinucleotide were used in steady-state kinetic studies with native and activated, amidinated enzymes. The Michaelis and inhibition constants increased up to 100-fold upon modification of coenzyme or enzyme. Turnover numbers with NAD+ and ethanol increased in some cases up to 10-fold due to increased rates of dissociation of enzyme-reduced coenzyme complexes. Rates of dissociation of oxidized coenzyme appeared to be mostly unaffected, but the values calculated (10-60 s-1) were significantly less than the turnover numbers with acetaldehyde and reduced coenzyme (20-900 s-1, at pH 8, 25 degrees C). Rates of association of coenzyme analogues also decreased up to 100-fold. When Lys-228 in the adenosine binding site was picolinimidylated, turnover numbers increased about 10-fold with NAD(H). Furthermore, the pH dependencies for association and dissociation of NAD+ and turnover number with NAD+ and ethanol showed the fastest rates above a pK value of 8.0. Turnover with NADH and acetaldehyde was fastest below a pK value of 8.1. These results can be explained by a mechanism in which isomerization of the enzyme-NAD+ complex (110 s-1) is partially rate limiting in turnover with NAD+ and ethanol (60 s-1) and is controlled by ionization of the hydrogen-bonded system that includes the water ligated to the catalytic zinc and the imidazole group of His-51.     

Alpha-isoenzyme of alcohol dehydrogenase from monkey liver. Cloning, expression, mechanism, coenzyme, and substrate specificity.

The cDNA for the alpha-isoenzyme from rhesus monkey (Macaca mulatta) liver was cloned and expressed in yeast. The alpha-isoenzymes of human and monkey liver alcohol dehydrogenase differ from the other human and horse liver enzymes in having Met57, Ala93, and Val116 instead of Leu57, Phe93, and Leu116 in the substrate binding pocket and Gly47 instead of Arg47 near the pyrophosphate moiety of the coenzyme. The effects of these differences on the kinetic mechanism, substrate specificity, and coenzyme binding were studied with the purified, recombinant monkey alpha-isoenzyme (MmADH alpha) and mutated enzymes with Gly47 substituted with His or Arg. The mechanism appears to be random for the binding of NAD+ and ethanol and ordered for NADH and acetaldehyde, with formation of a dead-end enzyme-NADH-ethanol complex. MmADH alpha reacts 130-fold slower (V/K) with ethanol and 3-25-fold slower with 2-methyl alcohols but 20-fold faster with cyclohexanol, as compared with horse (Equus caballus) liver EE isoenzyme (EqADH). MmADH alpha is stereoselective for the R isomer of 2-butanol, whereas EqADH favors the S isomer. Both enzymes have comparable reactivity with larger primary alcohols. MmADH alpha is more reactive with secondary alcohols and has highest activity with cyclohexanol. However, it does not react with steroids such as 5 beta-androstane-17 beta-ol-3-one. Molecular modeling suggests that the differences between MmADH alpha and EqADH are a result of the substitution of Ala for Phe93 and Thr for Ser48. MmADH alpha binds NAD+ most rapidly when a group with a pK of 7.4 is unprotonated, implicating His51 in this reaction. The G47R substitution decreased the dissociation constants for NAD+ and NADH and turnover numbers only about 2-fold, whereas the G47H substitution increased dissociation constants 7-14-fold and turnover numbers 4-fold. A basic residue at position 47 is not crucial for activity, as multiple interactions determine coenzyme affinity.     

The pH-dependent binding of NADH and subsequent enzyme isomerization of human liver beta 3 beta 3 alcohol dehydrogenase

Human class I beta 3 beta 3 is one of the alcohol dehydrogenase dimers that catalyzes the reversible oxidation of ethanol. The beta 3 subunit has a Cys substitution for Arg-369 (beta 369C) in the coenzyme-binding site of the beta1 subunit. Kinetic studies have demonstrated that this natural mutation in the coenzyme-binding site decreases affinity for NAD+ and NADH. Structural studies suggest that the enzyme isomerizes from an open to closed form with coenzyme binding. However, the extent to which this isomerization limits catalysis is not known. In this study, stopped-flow kinetics were used from pH 6 to 9 with recombinant beta 369C to evaluate rate-limiting steps in coenzyme association and catalysis. Association rates of NADH approached an apparent zero-order rate with increasing NADH concentrations at pH 7.5 (42 +/- 1 s-1). This observation is consistent with an NADH-induced isomerization of the enzyme from an open to closed conformation. The pH dependence of apparent zero-order rate constants fit best a model in which a single ionization limits diminishing rates (pKa = 7.2 +/- 0.1), and coincided with Vmax values for acetaldehyde reduction. This indicates that NADH-induced isomerization to a closed conformation may be rate-limiting for acetaldehyde reduction. The pH dependence of equilibrium NADH-binding constants fits best a model in which a single ionization leads to a loss in NADH affinity (pKa = 8.1 +/- 0. 2). Rate constants for isomerization from a closed to open conformation were also calculated, and these values coincided with Vmax for ethanol oxidation above pH 7.5. This suggests that NADH-induced isomerization of beta 369C from a closed to open conformation is rate-limiting for ethanol oxidation above pH 7.5.     

Contributions of valine-292 in the nicotinamide binding site of liver alcohol dehydrogenase and dynamics to catalysis

The participation of Val-292 in catalysis by alcohol dehydrogenase and the involvement of dynamics were investigated. Val-292 interacts with the nicotinamide ring of the bound coenzyme and may facilitate hydride transfer. The substitution of Val-292 with Ser (V292S) increases the dissociation constants for the coenzymes (NAD(+) by 50-fold, NADH by 75-fold) and the turnover numbers by 3-7-fold. The V292S enzyme crystallized in the presence of NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol has an open conformation similar to the structure of the wild-type apo-enzyme, rather than the closed conformation observed for ternary complexes with wild-type enzyme. The V292S substitution perturbs the conformational equilibrium of the enzyme and decreases the kinetic complexity, which permits study of the hydride transfer step with steady-state kinetics. Eyring plots show that the DeltaH for the oxidation (V(1)) of the protio and deuterio benzyl alcohols is 13 kcal/mol and that the kinetic isotope effect of 4.1 is essentially temperature-independent. Eyring plots for the catalytic efficiency for reduction of benzaldehyde (V(2)/K(p)) with NADH or NADD are distinctly convex, being temperature-dependent from 5 to 25 degrees C and temperature-independent from 25 to 50 degrees C; the kinetic isotope effect of 3.2 for V(2)/K(p) is essentially independent of the temperature. The temperature dependencies and isotope effects for V(1) and V(2)/K(p) are not adequately explained by semiclassical transition state theory and are better explained by hydride transfer occurring through vibrationally assisted tunneling.     

Expression and kinetic characterization of variants of human beta 1 beta 1 alcohol dehydrogenase containing substitutions at amino acid 47

Arg-47 of human beta 1 beta 1 alcohol dehydrogenase has been replaced with Lys, His, Gln, and Gly by site-directed mutagenesis. The mutated enzymes were expressed in Escherichia coli and purified to homogeneity. The recombinant enzymes with Arg and His at position 47 exhibit kinetic constants and stability which are similar to beta 1 beta 1 and beta 2 beta 2, respectively. The substitution of Lys, His, or Gln for Arg-47 resulted in active enzymes with lower affinity for coenzyme and higher Vmax values than beta 1 beta 1. The substitution of Gln at position 47 resulted in an enzyme with the highest Vmax for ethanol oxidation of any mammalian alcohol dehydrogenase. In this series of enzymes, the affinity for coenzyme decreases with decreasing pKa of the substituted amino acid side chains. The substitution of Gly at position 47 resulted in an enzyme with a Vmax that was one-half that of the low activity beta 1 beta 1 and coenzyme affinities that are lower than beta 1 beta 1, but are equal to or greater than the affinities exhibited by the His-47 or Gln-47 enzymes. Product inhibition studies indicated a change in mechanism from ordered Bi Bi for beta 1 beta 1 to rapid equilibrium random Bi Bi for the Gly-47 enzyme. The kinetic properties of the Gly-47 enzyme are substantially different from human liver alpha alpha which also has Gly at position 47.     

Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase

The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 ? resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.     

Kinetics of coenzyme binding to liver alcohol dehydrogenase in the pH range 10-12

On- and off-velocity constants for NADH and NAD+ binding to liver alcohol dehydrogenase in the pH range 10-12 have been determined by stopped-flow kinetic methods. The results are consistent with previously reported equilibrium binding data and proposals attributing the main effects of pH on coenzyme binding to ionization of Lys-228 and zinc-bound water. Deprotonation of the group identified as Lys-228 decreases the NADH and NAD+ association rates by a factor exceeding 20 and has no detectable effect on the coenzyme dissociation rates in the examined pH range. Ionization of the group identified as zinc-bound water causes a 3-fold increase of the rate of NADH dissociation from the enzyme, and decreases the rate of NAD+ dissociation by a factor of 200. The NADH and NAD+ association rates are decreased by a factor of 30 and 5, respectively. The observed effects of pH can be rationalized in terms of electrostatic interactions of the ionizing groups with the charges present on the coenzyme molecules and lend support to the idea that binding of the coenzyme nicotinamide ring occurs subsequent to binding of the AMP portion of the coenzyme.     

A study of the kinetics and mechanism of yeast alcohol dehydrogenase with a variety of substrates

1. The kinetics of oxidation of ethanol, propan-1-ol, butan-1-ol and propan-2-ol by NAD(+) and of reduction of acetaldehyde and butyraldehyde by NADH catalysed by yeast alcohol dehydrogenase were studied. 2. Results for the aldehyde-NADH reactions are consistent with a compulsory-order mechanism with the rate-limiting step being the dissociation of the product enzyme-NAD(+) complex. In contrast the results for the alcohol-NAD(+) reactions indicate that some dissociation of coenzyme from the active enzyme-NAD(+)-alcohol ternary complexes must occur and that the mechanism is not strictly compulsory-order. The rate-limiting step in ethanol oxidation is the dissociation of the product enzyme-NADH complex but with the other alcohols it is probably the catalytic interconversion of ternary complexes. 3. The rate constants describing the combination of NAD(+) and NADH with the enzyme and the dissociations of these coenzymes from binary complexes with the enzyme were measured.     

Control of coenzyme binding to horse liver alcohol dehydrogenase.

The rate of association of NAD(+) with wild-type horse liver alcohol dehydrogenase (ADH) is maximal at pH values between pK values of about 7 and 9, and the rate of NADH association is maximal at a pH below a pK of 9. The catalytic zinc-bound water, His-51 (which interacts with the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of the coenzyme in the proton relay system), and Lys-228 (which interacts with the adenosine 3'-hydroxyl group and the pyrophosphate of the coenzyme) may be responsible for the observed pK values. In this study, the Lys228Arg, His51Gln, and Lys228Arg/His51Gln (to isolate the effect of the catalytic zinc-bound water) mutations were used to test the roles of the residues in coenzyme binding. The steady state kinetic constants at pH 8 for the His51Gln enzyme are similar to those for wild-type ADH. The Lys228Arg and Lys228Arg/His51Gln substitutions decrease the affinity for the coenzymes up to 16-fold, probably due to altered interactions with the arginine at position 228. As determined by transient kinetics, the rate constant for association of NAD(+) with the mutated enzymes no longer decreases at high pH. The pH profile for the Lys228Arg enzyme retains the pK value near 7. The His51Gln and Lys228Arg/His51Gln substitutions significantly decrease the rate constants for NAD(+) association, and the pH dependencies show that these enzymes bind NAD(+) most rapidly at a pH above pK values of 8. 0 and 9.0, respectively. It appears that the pK of 7 in the wild-type enzyme is shifted up by the H51Q substitutions, and the resulting pH dependence is due to the deprotonation of the catalytic zinc-bound water. Kinetic simulations suggest that isomerization of the enzyme-NAD(+) complex is substantially altered by the mutations. In contrast, the pH dependencies for NADH association with His51Gln, Lys228Arg, and Lys228Arg/His51Gln enzymes were the same as for wild-type ADH, suggesting that the binding of NAD(+) and the binding of NADH are controlled differently.     

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.     

Mutation of amino acids thought to polarize the oxaloacetate carbonyl in citrate synthase severely reduces but does not abolish activity of the enzyme

Oligonucleotide-directed mutagenesis has been used to alter two active site residues of Escherichia coli citrate synthase, histidine-305 and arginine-314. Both residues are thought to be involved in the polarization of the carbonyl group of oxaloacetate and thus facilitate attack at the carbonyl carbon by acetyl-CoA. In one mutant, designated CS305H----A, His-305 was mutated to alanine and in the other, designated CS314R----L, Arg-314 was changed to leucine. Both mutants have greatly reduced turnover numbers, less than 0.1% of the wild-type value. The dissociation constant for formation of the binary enzyme-oxaloacetate complex, Ki, OAA, is at least 950 microM for CS305H----A, and about 500 microM for CS314R----L, 28 and 15 times the wild-type value, respectively. The Michaelis constants for the two substrates, KOAA and KAcCoA, which measure the affinity of the enzyme for the catalytically significant ternary complex, are less radically altered: values of KAcCoA are actually 3.5-fold and 4.6-fold lower for CS305H----A and CS314R----L, respectively. These kinetic effects are taken to mean that both His-305 and Arg-314 are important for the successful formation of an efficient transition state, very likely by polarizing the carbonyl group of oxaloacetate as has been suggested, and that the residual kinetic activity, in both mutants, occurs by a mechanism which benefits from only part of this polarization. Allosteric properties of the mutant enzymes, as measured by NADH inhibition and binding, and KCl activation, are normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substitutions in a flexible loop of horse liver alcohol dehydrogenase hinder the conformational change and unmask hydrogen transfer.

When horse liver alcohol dehydrogenase binds coenzyme, a rotation of about 10 degrees brings the catalytic domain closer to the coenzyme binding domain and closes the active site cleft. The conformational change requires that a flexible loop containing residues 293-298 in the coenzyme binding domain rearranges so that the coenzyme and some amino acid residues from the catalytic domain can be accommodated. The change appears to control the rate of dissociation of the coenzyme and to be necessary for installation of the proton relay system. In this study, directed mutagenesis produced the activated Gly293Ala/Pro295Thr enzyme. X-ray crystallography shows that the conformations of both free and complexed forms of the mutated enzyme and wild-type apoenzyme are very similar. Binding of NAD(+) and 2,2, 2-trifluoroethanol do not cause the conformational change, but the nicotinamide ribose moiety and alcohol are not in a fixed position. Although the Gly293Ala and Pro295Thr substitutions do not disturb the apoenzyme structure, molecular modeling shows that the new side chains cannot be accommodated in the closed native holoenzyme complex without steric alterations. The mutated enzyme may be active in the "open" conformation. The turnover numbers with ethanol and acetaldehyde increase 1.5- and 5.5-fold, respectively, and dissociation constants for coenzymes and other kinetic constants increase 40-2,000-fold compared to those of the native enzyme. Substrate deuterium isotope effects on the steady state V or V/K(m) parameters of 4-6 with ethanol or benzyl alcohol indicate that hydrogen transfer is a major rate-limiting step in catalysis. Steady state oxidation of benzyl alcohol is most rapid above a pK of about 9 for V and V/K(m) and is 2-fold faster in D(2)O than in H(2)O. The results are consistent with hydride transfer from a ground state zinc alkoxide that forms a low-barrier hydrogen bond with the hydroxyl group of Ser48.     

Transient kinetic studies of substrate inhibition in the horse liver alcohol dehydrogenase reaction

The rate-limiting step of ethanol oxidation by alcohol dehydrogenase (E) at substrate inhibitory conditions (greater than 500 mM ethanol) is shown to be the dissociation rate of NADH from the abortive E-ethanol-NADH complex. The dissociation rate constant of NADH decreased hyperbolically from 5.2 to 1.4 s-1 in the presence of ethanol causing a decrease in the Kd of NADH binding from 0.3 microM for the binary complex to 0.1 microM for the abortive complex. Correspondingly, ethanol binding to E-NADH (Kd = 37 mM) was tighter than to enzyme (Kd = 109 mM). The binding rate of NAD+ (7 X 10(5) M-1s-1) to enzyme was not affected by the presence of ethanol, further substantiating that substrate inhibition is totally due to a decrease in the dissociation rate constant of NADH from the abortive complex. Substrate inhibition was also observed with the coenzyme analog, APAD+, but a single transient was not found to be rate limiting. Nevertheless, the presence of substrate inhibition with APAD+ is ascribed to a decrease in the dissociation rate of APADH from 120 to 22 s-1 for the abortive complex. Studies to discern the additional limiting transient(s) in turnover with APAD+ and NAD+ were unsuccessful but showed that any isomerization of the enzyme-reduced coenzyme-aldehyde complex is not rate limiting. Chloride increases the rate of ethanol oxidation by hyperbolically increasing the dissociation rate constant of NADH from enzyme and the abortive complex to 12 and 2.8 s-1, respectively. The chloride effect is attributed to the binding of chloride to these complexes, destabilizing the binding of NADH while not affecting the binding of ethanol.     

Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.     

Enhancement of coenzyme binding by a single point mutation at the coenzyme binding domain of E. coli lactaldehyde dehydrogenase

Phenylacetaldehyde dehydrogenase (PAD) and lactaldehyde dehydrogenase (ALD) share some structural and kinetic properties. One difference is that PAD can use NAD+ and NADP+, whereas ALD only uses NAD+. An acidic residue has been involved in the exclusion of NADP+ from the active site in pyridine nucleotide-dependent dehydrogenases. However, other factors may participate in NADP+ exclusion. In the present work, analysis of the sequence of the region involved in coenzyme binding showed that residue F180 of ALD might participate in coenzyme specificity. Interestingly, F180T mutation rendered an enzyme (ALD-F180T) with the ability to use NADP+. This enzyme showed an activity of 0.87 micromol/(min * mg) and K(m) for NADP+ of 78 microM. Furthermore, ALD-F180T exhibited a 16-fold increase in the V(m) /K(m) ratio with NAD+ as the coenzyme, from 12.8 to 211. This increase in catalytic efficiency was due to a diminution in K(m) for NAD+ from 47 to 7 microM and a higher V(m) from 0.51 to 1.48 micromol/(min * mg). In addition, an increased K(d) for NADH from 175 (wild-type) to 460 microM (mutant) indicates a faster product release and possibly a change in the rate-limiting step. For wild-type ALD it is described that the rate-limiting step is shared between deacylation and coenzyme dissociation. In contrast, in the present report the rate-limiting step in ALD-F180T was determined to be exclusively deacylation. In conclusion, residue F180 participates in the exclusion of NADP+ from the coenzyme binding site and disturbs the binding of NAD+.     

The AdhS alleloenzyme of alcohol dehydrogenase from Drosophila melanogaster. Variation of kinetic parameters with pH.

The variation with pH of the kinetic parameters for the alcohol and acetaldehyde reactions were studied for the alleloenzyme AdhS from Drosophila melanogaster. The variation of Ki (KEO,I) with pH for two ethanol-competitive inhibitors, pyrazole and 2,2,2-trifluoroethanol, was also studied. Both alcohol oxidation and acetaldehyde reduction follow a compulsory ordered pathway, with coenzyme binding first. The rate-limiting step for ethanol oxidation is complex and involves at least hydride transfer and dissociation of the enzyme-NADH complex (ER). In contrast with this, the rate-limiting step for the back reaction, i.e. the reduction of acetaldehyde, is dissociation of the enzyme-NAD+ complex (EO). A rate-limiting ER dissociation appears in the oxidation of the secondary alcohol propan-2-ol, whereas for the back reaction, i.e. acetone reduction, hydride transfer in the ternary complexes is rate-limiting. There is one group in the free enzyme, with a pK of approx. 8.0, that regulates the kon velocity for NADH, whereas for NAD+ several groups seem to be involved. A group in the enzyme is drastically perturbed by the formation of the binary EO complex. Protonation of this group with a pK of 7.6 in the EO complex resulted in weakened alcohol and inhibitor binding, in addition to an increased dissociation rate of NAD+ from the binary EO complex. Neither the binding of acetaldehyde nor the dissociation rate of NADH from the binary ER complex varied within the pH region studied.     

An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme

In the three-dimensional structures of enzymes that bind NAD or FAD, there is an acidic residue that interacts with the 2'- and 3'-hydroxyl groups of the adenosine ribose of the coenzyme. The size and charge of the carboxylate might repel the binding of the 2'-phosphate group of NADP and explain the specificity for NAD. In the NAD-dependent alcohol dehydrogenases, Asp-223 (horse liver alcohol dehydrogenase sequence) appears to have this role. The homologous residue in yeast alcohol dehydrogenase I (residue 201 in the protein sequence) was substituted with Gly, and the D223G enzyme was expressed in yeast, purified, and characterized. The wild-type enzyme is specific for NAD. In contrast, the D223G enzyme bound and reduced NAD+ and NADP+ equally well, but, relative to wild-type enzyme, the dissociation constant for NAD+ was increased 17-fold, and the reactivity (V/K) on ethanol was decreased to 1%. Even though catalytic efficiency was reduced, yeast expressing the altered or wild-type enzyme grew at comparable rates, suggesting that equilibration of NAD and NADP pools is not lethal. Asp-223 participates in binding NAD and in excluding NADP, but it is not the only residue important for determining specificity for coenzyme.     

Interaction of alcohol dehydrogenase with tert-butylhydroperoxide: stimulation of the horse liver and inhibition of the yeast enzymes

Preincubation of horse liver alcohol dehydrogenase (HLADH) with the oxidative agent, tert-butyl hydroperoxide (tBOOH) results in a twofold stimulation of the ethanol dehydrogenase activity of this enzyme. This stimulation was dependent on tBOOH concentration up to 100 mM; above this concentration tBOOH did not further stimulate ethanol oxidation by HLADH. Active-site-directed reagents and classical ADH binary complexes were used to probe the possible mechanism of this activating effect. The rate and extent of stimulation by tBOOH is strongly reduced by binary complexes with NAD(+) or NADH, whose pyrophosphate groups bind to Arg-47 and Arg-369. In contrast stimulation by tBOOH was not prevented by AMP or the sulfhydryl reagents dithiothreitol and glutathione, suggesting, respectively, a lack of role for Lys-228 and sulfhydryl group oxidation in the stimulation by tBOOH. In contrast to the liver enzyme, treatment of yeast ADH (YADH) with tBOOH irreversibly inhibited its ethanol dehydrogenase activity. Inhibition of YADH by tBOOH approximated first-order rate kinetics with respect to enzyme at fixed concentrations of tBOOH between 0.5 to 300 mM. Four -SH groups per molecule of YADH were modified by tBOOH, whereas only two -SH groups were modified in HLADH. The stimulation of HLADH by tBOOH is suggested to be due to destabilization of the catalytic Zn-coordination sphere and amino acids associated with coenzyme binding in the active site, while inactivation of YADH appears to be associated with -SH group oxidation by the peroxide.     

Structural and functional properties of aldose xylose reductase from the D-xylose-metabolizing yeast Candida tenuis

The primary structure of the aldose xylose reductase from Candida tenuis (CtAR) is shown to be 39% identical to that of human aldose reductase (hAR). The catalytic tetrad of hAR is completely conserved in CtAR (Tyr51, Lys80, Asp46, His113). The amino acid residues involved in binding of NADPH by hAR (D.K. Wilson, et al., Science 257 (1992) 81-84) are 64% identical in CtAR. Like hAR the yeast enzyme is specific for transferring the 4-pro-R hydrogen of the coenzyme. These properties suggest that CtAR is a member of the aldo/keto reductase superfamily. Unlike hAR the enzyme from C. tenuis has a dual coenzyme specificity and shows similar specificity constants for NADPH and NADH. It binds NADP(+) approximately 250 times less tightly than hAR. Typical turnover numbers for aldehyde reduction by CtAR (15-20 s(-1)) are up to 100-fold higher than corresponding values for hAR, probably reflecting an overall faster dissociation of NAD(P)(+) in the reaction catalyzed by the yeast enzyme.