PubMed Health⌕ Search

Biomedical subjects

B V Plapp

Publications and source records attributed to B V Plapp.

At least 37 records · Page 2Linked to original sources
PubMed1994

Crystallization and preliminary crystallographic studies of Saccharomyces cerevisiae alcohol dehydrogenase I.

The cytoplasmic yeast alcohol dehydrogenase I crystallized at 5 degrees C as hexagonal plates or short columns in the presence of NAD+ and 2,2,2-trifluoroethanol, in sodium N-tris(hydroxymethyl)methyl-3-aminopropanesulfonate buffer at pH 8.2 to 8.6, using polyethylene glycol 4000 as precipitant. X-ray diffraction data to 3.2 A resolution show that the crystals are hexagonal in space group P622 with unit cell dimensions a = b = 147.9 A, c = 69.1 A. There is one subunit of the tetrameric enzyme per asymmetric unit, giving a packing density of 2.9 A3/Da.

Alcohol Dehydrogenase↗
PubMed1994

Crystallographic investigations of alcohol dehydrogenases.

The structures of horse liver alcohol dehydrogenase class I in its apoenzyme form and in different ternary complexes have been determined at high resolution. The complex with NAD+ and the substrate analogue pentafluorobenzyl alcohol gives a detailed picture of the interactions in an enzyme-substrate complex. The alcohol is bound to the zinc and positioned so that the hydrogen atom can be directly transferred to the C4 atom of the nicotinamide ring. The structure of cod liver alcohol dehydrogenase with hybrid properties (functionally of class I but structurally overall closer to class III) has been determined by molecular replacement methods to 3 A resolution. Yeast alcohol dehydrogenase has been crystallized, and native data have been collected to 3 A resolution.

Alcohol Dehydrogenase↗
PubMed1994

Control of alcohol metabolism.

The rate of alcohol metabolism is determined by the kinetic characteristics and concentrations of the alcohol and aldehyde dehydrogenases and by the rate of restoration of the redox state of the cell. Several potent competitive and uncompetitive inhibitors of the alcohol dehydrogenases can decrease the rate of alcohol metabolism; they may be useful for preventing the potentially deleterious effects of ethanol metabolism. Alcohol dehydrogenases have very broad specificity and can readily reduce a variety of carbonyl compounds by exchange reactions while ethanol is metabolized. Agents that increase the rate of metabolism need to be developed.

Alcohol Dehydrogenase↗
PubMed1993

Alternative pathways and reactions of benzyl alcohol and benzaldehyde with horse liver alcohol dehydrogenase.

Liver alcohol dehydrogenase catalyzes the reaction of NAD+ and benzyl alcohol to form NADH and benzaldehyde by a predominantly ordered reaction. However, enzyme-alcohol binary and abortive ternary complexes form at high concentrations of benzyl alcohol, and benzaldehyde is slowly oxidized to benzoic acid. Steady-state and transient kinetic studies, equilibrium spectrophotometric measurements, product analysis, and kinetic simulations provide estimates of rate constants for a complete mechanism with the following reactions: (1) E<-->E-NAD+<-->E-NAD(+)-RCH2OH<-->E-NADH-RCHO<-->E-NADH<-->E ; (2) E-NADH<-->E-NADH-RCH2OH<-->E-RCH2OH<-->E; (3) E-NAD+<-->E-NAD(+)-RCHO-->E- NADH-RCOOH<-->E-NADH. The internal equilibrium constant for hydrogen transfer determined at 30 degrees C and pH 7 is about 5:1 in favor of E-NAD(+)-RCH2OH and has a complex pH dependence. Benzyl alcohol binds weakly to free enzyme (Kd = 7 mM) and significantly decreases the rates of binding of NAD+ and NADH. The reaction of NAD+ and benzyl alcohol is therefore kinetically ordered, not random. High concentrations of benzyl alcohol (> 1 mM) inhibit turnover by formation of the abortive E-NADH-RCH2-OH complex, which dissociates at 0.3 s-1 as compared to 6.3 s-1 for E-NADH. The oxidation of benzaldehyde by E-NAD+ (Km = 15 mM, V/E = 0.4 s-1) is inefficient relative to the oxidation of benzyl alcohol (Km = 28 microM, V/E = 3.1 s-1) and leads to a dismutation (2RCHO-->RCH2OH + RCOOH) as E-NADH reduces benzaldehyde. The results provide a description of final product distributions for the alternative reactions catalyzed by the multifunctional enzyme.

Alcohol Dehydrogenase↗
PubMed1993

Unmasking of hydrogen tunneling in the horse liver alcohol dehydrogenase reaction by site-directed mutagenesis.

Primary and secondary kD/kT and kH/kT kinetic isotope effects have been studied as a probe of hydrogen tunneling in the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase (LADH). In the case of the wild-type enzyme, isotope effects at 25 degrees C do not clearly support hydrogen tunneling; this result is consistent with a reaction rate that is partially limited by the release of product benzaldehyde. The three-dimensional structure for LADH was used to design site-directed mutations in an effort to enhance the rate of the product release step and to "unmask" tunneling. Substitutions that increased the size of the alcohol binding pocket resulted in minor changes in isotope effects. By contrast, reduction in the size of the alcohol binding pocket through substitution at residues 57 and 93, which are in van der Waals contact with bound alcohol substrate, produced a clear demonstration of protium tunneling from the breakdown of the semiclassical relationship between kD/kT and kH/kT isotope effects. The temperature dependence of kD/kT isotope effects has also been pursued, leading to the conclusion that tunneling does, in fact, occur in the reaction catalyzed by wild-type LADH. Despite the unmasking of protium tunneling in site-directed mutants, substitutions that decrease the size of the alcohol pocket appear to result in less extensive tunneling in the hydride transfer. It is noteworthy that the mutant enzyme (Leu57-->Phe), which shows the greatest evidence of tunneling, has the same catalytic efficiency (Vmax/Km) as the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1993

Inversion of the substrate specificity of yeast alcohol dehydrogenase.

The relationship between the size of the substrate binding pocket and the catalytic reactivities with varied alcohols was studied with the Saccharomyces cerevisiae alcohol dehydrogenase I (ScADH) and compared with the liver enzymes from horse (EqADH, EE isoenzyme) and monkey (MmADH alpha, alpha-isoenzyme). The yeast enzyme is most active with ethanol, and its activity decreases as the size of the alcohol is increased, whereas the activities of the liver enzymes increase with larger alcohols. The substrate pocket in ScADH was enlarged by single substitutions of Thr-48 to Ser (T48S), Trp-57 to Met (W57M), and Trp-93 to Ala (W93A), and a double change, T48S:W93A, and a triple, T48S:W57M:W93A. The T48S enzyme has the same pattern of activity (V/K) as wild-type ScADH for linear primary alcohols. The W57M enzymes have lowered reactivity with primary and secondary alcohols. The W93A and T48S:W93A enzymes resemble MmADH alpha in having an inverted specificity pattern for primary alcohols, being 3- and 10-fold more active on hexanol and 350- and 540-fold less active on ethanol, and are as reactive as the liver enzymes with long chain primary alcohols. The three Ala-93 enzymes also acquired weak activity on branched chain alcohols and cyclohexanol.

Aldehyde Dehydrogenase↗
PubMed1992

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.

Alcohol Dehydrogenase↗
PubMed1992

Interconversion of E and S isoenzymes of horse liver alcohol dehydrogenase. Several residues contribute indirectly to catalysis.

The E and S isoenzymes of horse liver alcohol dehydrogenase differ by 10 amino acid residues, but only the S isoenzyme is active on 3 beta-hydroxysteroids. This functional difference was correlated to the differences in structures of the isoenzymes by characterizing a series of chimeric enzymes, which could represent intermediates in the evolution of catalytic activity. Deletion of Asp-115 from the E isoenzyme created the E/D115 delta enzyme that is active on steroids. The deletion alters the substrate binding pocket by moving Leu-116, which sterically hinders binding of steroids in the E isoenzyme. A chimeric enzyme (ESE) that has four changes in or near the substrate binding pocket (T94I/R101S/F110L/D115 delta) was 15-30-fold more catalytically efficient (V/Km) on uncharged steroids than was the E/D115 delta enzyme. Molecular modeling suggests that the substitutions at residues 94 and 110 indirectly affect the activity on steroids. ESE enzyme was 6-fold more active than the S isoenzyme on neutral steroids, due to substitutions not in the substrate binding pocket. The K366E and the Q17E/A43T/A59T substitutions in the S isoenzyme gave 2-fold increases in V/Km on steroids, which together can account for the changes observed with the ESE enzyme. The enzymes that are active on steroids did not bind 2,2,2-trifluoroethanol as tightly and were catalytically less efficient than the E isoenzyme with small alcohols. However, these enzymes were two to three and four to five orders of magnitude more efficient with 1-hexanol and 5 beta-androstane-3 beta,17 beta-diol, respectively, than with ethanol. These results demonstrate that several residues not directly participating in substrate binding or chemical catalysis contribute to catalytic efficiency.

Alcohol Dehydrogenase↗
PubMed1992

Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family.

Sequences of 47 members of the Zn-containing alcohol dehydrogenase (ADH) family were aligned progressively, and an evolutionary tree with detailed branch order and branch lengths was produced. The alignment shows that only 9 amino acid residues (of 374 in the horse liver ADH sequence) are conserved in this family; these include eight Gly and one Val with structural roles. Three residues that bind the catalytic Zn and modulate its electrostatic environment are conserved in 45 members. Asp 223, which determines specificity for NAD, is found in all but the two NADP-dependent enzymes, which have Gly or Ala. Ser or Thr 48, which makes a hydrogen bond to the substrate, is present in 46 members. The four Cys ligands for the structural zinc are conserved except in zeta-crystallin, the sorbitol dehydrogenases, and two bacterial enzymes. Analysis of the evolutionary tree gives estimates of the times of divergence for different animal ADHs. The human class II (pi) and class III (chi) ADHs probably diverged about 630 million years ago, and the newly identified human ADH6 appeared about 520 million years ago, implying that these classes of enzymes may exist or have existed in all vertebrates. The human class I ADH isoenzymes (alpha, beta, and gamma) diverged about 80 million years ago, suggesting that these isoenzymes may exist or have existed in all primates. Analysis of branch lengths shows that these plant ADHs are more conserved than the animal ones and that class III ADHs are more conserved than class I ADHs. The rate of acceptance of point mutations (PAM units) shows that selection pressure has existed for ADHs, implying that these enzymes play definite metabolic roles.

Alcohol Dehydrogenase↗
PubMed1991

Isoenzymes of horse liver alcohol dehydrogenase active on ethanol and steroids. cDNA cloning, expression, and comparison of active sites.

Horse liver alcohol dehydrogenase occurs as isoenzymes: E is active on ethanol but not steroids; S is active on ethanol and steroids. The cDNAs for these isoenzymes were cloned; both were 1.8-kilobase long and contained complete coding sequences. Both enzymes were expressed in Escherichia coli, and the purified proteins had properties similar to those of the natural enzymes. The amino acid sequence deduced from the open reading frame of the E-type cDNA agreed with the amino acid sequence of the E isoenzyme determined by protein sequencing and x-ray crystallography. When compared with the E-type cDNA, the coding region of the S-type cDNA contains 24 substitutions and 3 deletions, giving rise to an amino acid sequence for the S. isoenzyme that differs from that of the E isoenzyme at 10 positions: nine conservative substitutions and one deletion, of Asp-115. These changes can be accommodated in the three-dimensional structure of the E isoenzyme, and models of the E and S isoenzymes complexed with a 3 beta-hydroxy-5 beta-steroid were built. The modeling shows that Leu-116 apparently sterically hinders binding of steroids in the E isoenzyme, and deletion in the S isoenzyme of Asp-115 moves Leu-116 and relieves the hindrance. The human gamma and rat liver enzymes are also active on steroids, but they have a different constellation of amino acid residues in the substrate pocket. Thus, there are multiple bases for the activity on steroids.

Alcohol Dehydrogenase↗
PubMed1991

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.

Alcohol Dehydrogenase↗
PubMed1991

Catalysis by yeast alcohol dehydrogenase.

Table 7 presents a brief summary of the effects of various mutations on some of the relevant kinetic constants. The results illustrate several important features of the use of site-directed mutagenesis in exploring structure and function of enzymes. Note that most of the mutations affect a given step or kinetic parameter in the mechanism, such as the binding of NAD+ or the turnover number with ethanol. Furthermore, one mutation can affect many steps in the mechanism. Thus, it is difficult to ascribe a particular role to an amino acid residue. It is also difficult to quantify the function of a residue, since the magnitudes of the effects on kinetic parameters will be modulated by the other amino acid residues that participate in the reaction. Comprehensive and quantitative kinetic studies of many mutant enzymes are required if we are to understand catalysis and specificity. We are reluctant to describe any residue as "essential" for activity since substitution with some amino acid can probably produce an enzyme with some residual activity. (Maybe the Thr48Gly enzyme would be active, as a water molecule could substitute for the hydroxyl of the threonine.) Likewise, when substitution of a residue partially, but not totally, decreases activity, it does not necessarily mean that the residue is "not essential". The change in activity can reflect the contribution of that residue to catalysis. On the other hand, if various substitutions of a residue do not change activity, it would be reasonable to conclude that the residue is not essential (Plapp et al., 1971). Most of the amino acid residues at the active site are involved in the catalytic mechanism, either by contacting the substrates directly or by participating in the chemistry. Some of the residues that are outside of the active site are indirectly involved, by affecting the structure of the protein. Substitution of an important amino acid residue should significantly affect activity, and studies on the kinetics and structure should allow one to distinguish among the various explanations.

Alcohol Dehydrogenase↗
PubMed1990

Substitution of arginine for histidine-47 in the coenzyme binding site of yeast alcohol dehydrogenase I.

Molecular modeling of alcohol dehydrogenase suggests that His-47 in the yeast enzyme (His-44 in the protein sequence, corresponding to Arg-47 in the horse liver enzyme) binds the pyrophosphate of the NAD coenzyme. His-47 in the Saccharomyces cerevisiae isoenzyme I was substituted with an arginine by a directed mutation. Steady-state kinetic results at pH 7.3 and 30 degrees C of the mutant and wild-type enzymes were consistent with an ordered Bi-Bi mechanism. The substitution decreased dissociation constants by 4-fold for NAD+ and 2-fold for NADH while turnover numbers were decreased by 4-fold for ethanol oxidation and 6-fold for acetaldehyde reduction. The magnitudes of these effects are smaller than those found for the same mutation in the human liver beta enzyme, suggesting that other amino acid residues in the active site modulate the effects of the substitution. The pH dependencies of dissociation constants and other kinetic constants were similar in the two yeast enzymes. Thus, it appears that His-47 is not solely responsible for a pK value near 7 that controls activity and coenzyme binding rates in the wild-type enzyme. The small substrate deuterium isotope effect above pH 7 and the single exponential phase of NADH production during the transient oxidation of ethanol by the Arg-47 enzyme suggest that the mutation makes an isomerization of the enzyme-NAD+ complex limiting for turnover with ethanol.

Alcohol Dehydrogenase↗
PubMed1990

Rate constants for a mechanism including intermediates in the interconversion of ternary complexes by horse liver alcohol dehydrogenase.

Transient kinetic data for partial reactions of alcohol dehydrogenase and simulations of progress curves have led to estimates of rate constants for the following mechanism, at pH 8.0 and 25 degrees C: E in equilibrium E-NAD+ in equilibrium *E-NAD+ in equilibrium E-NAD(+)-RCH2OH in equilibrium E-NAD+-RCH2O- in equilibrium *E-NADH-RCHO in equilibrium E-NADH-RCHO in equilibrium E-NADH in equilibrium E. Previous results show that the E-NAD+ complex isomerizes with a forward rate constant of 620 s-1 [Sekhar, V. C., & Plapp, B. V. (1988) Biochemistry 27, 5082-5088]. The enzyme-NAD(+)-alcohol complex has a pK value of 7.2 and loses a proton rapidly (greater than 1000 s-1). The transient oxidation of ethanol is 2-fold faster in D2O, and proton inventory results suggest that the transition state has a charge of -0.3 on the substrate oxygen. Rate constants for hydride ion transfer in the forward or reverse reactions were similar for short-chain aliphatic substrates (400-600 s-1). A small deuterium isotope effect for transient oxidation of longer chain alcohols is apparently due to the isomerization of the E-NAD+ complex. The transient reduction of aliphatic aldehydes showed no primary deuterium isotope effect; thus, an isomerization of the E-NADH-aldehyde complex is postulated, as isomerization of the E-NADH complex was too fast to be detected. The estimated microscopic rate constants show that the observed transient reactions are controlled by multiple steps.

Alcohol Dehydrogenase↗
PubMed1988

Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide.

The binding of NAD+ to liver alcohol dehydrogenase was studied by stopped-flow techniques in the pH range from 6.1 to 10.9 at 25 degrees C. Varying the concentrations of NAD+ and a substrate analogue used to trap the enzyme-NAD+ complex gave saturation kinetics. The same maximum rate constants were obtained with or without the trapping agent and by following the reaction with protein fluorescence or absorbance of a ternary complex. The data fit a mechanism with diffusion-controlled association of enzyme and NAD+, followed by an isomerization with a forward rate constant of 500 s-1 at pH 8: E E-NAD+ *E-NAD+. The isomerization may be related to the conformational change determined by X-ray crystallography of free enzyme and enzyme-coenzyme complexes. Overall bimolecular rate constants for NAD+ binding show a bell-shaped pH dependence with apparent pK values at 6.9 and 9.0. Acetimidylation of epsilon-amino groups shifts the upper pK to a value of 11 or higher, suggesting that Lys-228 is responsible for the pK of 9.0. Formation of the enzyme-imidazole complex abolishes the pK value of 6.9, suggesting that a hydrogen-bonded system extending from the zinc-bound water to His-51 is responsible for this pK value. The rates of isomerization of E-NAD+ and of pyrazole binding were maximal at pH below a pK of about 8, which is attributable to the hydrogen-bonded system. Acetimidylation of lysines or displacement of zinc-water with imidazole had little effect on the rate of isomerization of the E-NAD+ complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1988

Reactivity of horse liver alcohol dehydrogenase with 3-methylcyclohexanols.

The specificity of horse liver alcohol dehydrogenase for cyclohexanol and its 3-methyl derivatives was investigated by stopped-flow and initial velocity kinetic studies. The (1S,3S)-3-methylcyclohexanol was 7 times more reactive (V/Km) than cyclohexanol, whereas the (1R,3R)-3-methylcyclohexanol was at least 1000 times less reactive than its enantiomer. Computer simulation of the transient reaction of NAD+ and the cyclohexanols catalyzed by the enzyme suggests that the rate of transfer of hydrogen from the alcohol to NAD+ is increased with the 1S,3S isomer. Modeling of the three-dimensional structure of the ternary complex of the enzyme suggests that the 1S,3S isomer should only bind in a productive, reactive mode, whereas the 1R,3R isomer would bind predominantly in a nonproductive, inhibitory mode.

Alcohol Dehydrogenase↗
PubMed1988

Carboxyl groups near the active site zinc contribute to catalysis in yeast alcohol dehydrogenase.

The importance of carboxyl groups near the active site zinc for the catalytic function of alcohol dehydrogenase I from Saccharomyces cerevisiae was examined by directed mutagenesis and steady state kinetics. Asp-49 was changed to asparagine and Glu-68 to glutamine (residue numbering as for horse liver enzyme). The catalytic efficiencies (V/Km) for ethanol oxidation and acetaldehyde reduction were decreased by factors of 1000 with the Asn-49 mutant and 100 with the Gln-68 enzyme. For the Asn-49 mutant, dissociation constants for coenzymes increased 7-fold, and Michaelis and inhibition constants for substrates and substrate analogs increased by factors of 20-50. The turnover numbers were reduced 50-fold for ethanol oxidation and 15-fold for acetaldehyde reduction. Product and dead-end inhibition studies and kinetic isotope effects showed that the mechanism with NAD+ and ethanol was rapid equilibrium random, in contrast to the ordered mechanism of wild-type enzyme. Alcohol dehydrogenase I and the Asn-49 mutant had similar CD spectra and 2 zinc atoms/subunit, but slightly different UV absorption and fluorescence spectra. The Gln-68 mutant resembled the wild-type enzyme in most kinetic constants, but the turnover number for ethanol oxidation decreased 35-fold, and Kd for NAD+ and Km for acetaldehyde increased by factors of 4 and 50, respectively. The pK values for V1 and V1/Km for ethanol oxidation were shifted from 7.7 (wild-type) to 6.8 in the Gln-68 and 6.2 in the Asn-49 mutant. The altered electrostatic environment near the active site zinc apparently decreases activities by hindering isomerizations of enzyme-substrate complexes.

Alcohol Dehydrogenase↗