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B V Plapp

Publications and source records attributed to B V Plapp.

At least 55 records · Page 3Linked to original sources
PubMed1987

Kinetic characterization of yeast alcohol dehydrogenases. Amino acid residue 294 and substrate specificity.

A three-dimensional model of yeast alcohol dehydrogenase, based on the homologous horse liver enzyme, was used to compare the substrate binding pockets of the three isozymes (I, II, and III) from Saccharomyces cerevisiae and the enzyme from Schizosaccharomyces pombe. Isozyme I and the S. pombe enzyme have methionine at position 294 (numbered as in the liver enzyme, corresponding to 270 in yeast), whereas isozymes II and III have leucine. Otherwise the active sites of the S. cerevisiae enzymes are the same. All four wild-type enzymes were produced from the cloned genes. In addition, oligonucleotide-directed mutagenesis was used to change Met-294 in alcohol dehydrogenase I to leucine. The mechanisms for all five enzymes were predominantly ordered with ethanol (but partially random with butanol) at pH 7.3 and 30 degrees C. The wild-type alcohol dehydrogenases and the leucine mutant had similar kinetic constants, except that isozyme II had 10-20-fold smaller Michaelis and inhibition constants for ethanol. Thus, residue 294 is not responsible for this difference. Apparently, substitutions outside of the substrate binding pocket indirectly affect the interactions of the alcohol dehydrogenases with ethanol. Nevertheless, the substitution of methionine with leucine in the substrate binding site of alcohol dehydrogenase I produced a 7-10-fold increase in reactivity (V/Km) with butanol, pentanol, and hexanol. The higher activity is due to tighter binding of the longer chain alcohols and to more rapid hydrogen transfer.

Alcohol Oxidoreductases↗
PubMed1987

Structure and function in yeast alcohol dehydrogenases.

The structure and kinetics of the isozymes from Saccharomyces cerevisiae (ADH I, II, III) have been compared, and the ADH I gene was specifically mutagenized in order to substitute amino acid residues of particular interest. A model of the yeast enzyme was constructed on the basis of the structure of the homologous horse liver enzyme. Steady state kinetic studies, at pH 7.3 and 30 degrees C, showed that the enzymes follow the Ordered Bi Bi mechanism. ADH II has a Michaelis constant for ethanol that is 10-fold smaller than the constants found for ADH I or III. Replacement of Met-294 (liver numbering) in the substrate binding pocket of ADH I with Leu, as found in ADH II, could be responsible for the different kinetics. However, the mutant enzyme, ADH I-Leu, had constants with ethanol that were similar to those of ADH I. Nevertheless, the Leu enzyme had better catalytic activity with longer chain alcohols than did the Met enzyme. Other substitutions must account for the differences between ADH I and II. His-47 binds the pyrophosphate of coenzyme, and replacement with Arg (as in the liver enzyme) decreases turnover numbers by 6-fold and dissociation constants for NAD+ and NADH by only 2 to 4-fold. The lower turnover number explains why yeast harboring the mutant Arg enzyme are resistant to poisoning by allyl alcohol. ADH I and ADH I-Arg have maximal activity on ethanol at pH values above a pK of about 7. Replacement of His-51 with Gln reduces activity 12-fold and abolishes the pK value.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1987

Characterization of alcohol dehydrogenase from cultured rat hepatoma (HTC) cells.

The highly active alcohol dehydrogenase (EC 1.1.1.1) in rat hepatic tumor cells (HTC) was purified 120-fold by chromatography on DEAE-Sepharose and AMP-Agarose to yield an enzyme with a specific activity of 88 mumole/min/mg protein, assayed with 1.7 mM NAD+ and 0.55 M ethanol at pH 9 and 30 degrees C. (By comparison, purified, normal rat liver enzyme has an activity of about 1 unit/mg.) Based on its physical and kinetic properties, we conclude that the HTC isozyme is the same as the enzyme from rat stomach and another rat hepatoma (Cederbaum AI, Pietruszko R, Hempel J, Becker FF, and Rubin E (1975) Arch Biochem Biophys 171:348-360). The kinetics of the HTC enzyme are consistent with the Ordered Bi Bi mechanism. The kinetic constants are generally much larger for the HTC enzyme than for the normal rat liver enzyme. The Michaelis constants for ethanol and acetaldehyde (Kb = 1100 mM, Kp = 260 mM) are 1000-fold larger, and the constants for NADH are 10 to 50-fold larger. Although the HTC enzyme has low catalytic efficiency (V/Kb) on ethanol, it has much better activity on longer chain alcohols, but no activity on cyclohexanol. The pH dependence of V/Kb with ethanol is unusual in that it appears to be a linear function of pH, increasing with a slope of 0.56. Thus, the active sites of the liver and HTC enzymes may be different, although the HTC enzyme is inactivated by bromoacetate and bipyridine as is found for the liver enzyme. The HTC (stomach) enzyme may function to oxidize high concentrations of ingested ethanol or longer chain alcohols.

Alcohol Dehydrogenase↗
PubMed1986

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.

Alcohol Dehydrogenase↗
PubMed1985

Inhibition of liver alcohol dehydrogenase and ethanol metabolism by 3-substituted thiolane 1-oxides.

3-Substituted thiolane 1-oxides (methyl, n-butyl, n-hexyl, and phenyl) were prepared and tested as inhibitors of horse, monkey, and rat liver alcohol dehydrogenases and of ethanol metabolism in rats. These compounds inhibit alcohol oxidation in an uncompetitive manner with respect to ethanol as a varied substrate. Lengthening the alkyl substituent increased the inhibitory potency because of tighter binding in the hydrophobic substrate binding pocket of the alcohol dehydrogenases. Thus, the 3-hexyl derivative was the most potent inhibitor of the purified rat liver alcohol dehydrogenase, with a Kii value of 0.13 microM. The 3-butyl derivative was the best inhibitor of ethanol metabolism in rats, with a Kii value of 11 mumol/kg. The acute toxicity in mice of the butyl derivative was 1.4 mmol/kg. Since high concentrations of alcohol do not prevent the inhibitory effects of these compounds, they may be particularly useful for preventing poisoning by methanol or ethylene glycol.

Alcohol Dehydrogenase↗
PubMed1984

Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with 3-bromopropionic acid.

Horse liver alcohol dehydrogenase is inactivated with Michaelis kinetics at pH 7 and 25 degrees C by 3-bromopropionic acid. In the absence of NAD+, the Ki is 2 mM, and the pseudo bimolecular rate constant (k3/Ki) is 0.03 M-1 s-1; in the presence of 1 mM NAD+, Ki is 2.3 mM, and k3/Ki is 0.006 M-1 s-1. 3-Bromopropionic acid is a competitive inhibitor, Ki of 0.4 mM, against ethanol as a substrate. Inactivation was prevented in the ternary complexes with NAD+ X pyrazole and NADH X isobutyramide, was retarded by NAD+, NADH, or bipyridine, and was almost unaffected by imidazole and AMP. Carboxyethylated enzyme did not detectably (as observed spectrophotometrically) bind bipyridine, NAD+, or NADH. Enzyme was inactivated with radioactive 3-bromopropionic acid, aminoethylated, and digested with trypsin and chymotrypsin. Analysis of the labeled peptides showed that Cys-174 was predominantly modified. In the presence of 1 mM NAD+, the reaction was much less specific. The interaction of the carboxyl group of 3-bromopropionic acid with the guanidino group of Arg-369 probably facilitates the selective reaction with Cys-174, which is ligated to the zinc at the active site. Carboxyethylation apparently inactivates by interfering with the proper binding of the pyrophosphate of the coenzyme to the enzyme.

Alcohol Dehydrogenase↗
PubMed1984

Quantitative analysis of lysine analogs and derivatives.

Methods for the modification of lysine residues in proteins and the analysis of artificially or naturally modified lysine derivatives by quantitative chromatographic procedures are described. The compilation of results should assist structure-function studies and the analysis of new lysine derivatives.

Amino Acids↗
PubMed1984

Kinetics of inhibition of ethanol metabolism in rats and the rate-limiting role of alcohol dehydrogenase.

If liver alcohol dehydrogenase were rate-limiting in ethanol metabolism, inhibitors of the enzyme should inhibit the metabolism with the same type of kinetics and the same kinetic constants in vitro and in vivo. Against varied concentrations of ethanol, 4-methylpyrazole is a competitive inhibitor of purified rat liver alcohol dehydrogenase (Kis = 0.11 microM, in 83 mM potassium phosphate and 40 mM KCl buffer, pH 7.3, 37 degrees C) and is competitive in rats (with Kis = 1.4 mumol/kg). Isobutyramide is essentially an uncompetitive inhibitor of purified enzyme (Kii = 0.33 mM) and of metabolism in vivo (Kii = 1.0 mmol/kg). Low concentrations of both inhibitors decreased the rate of metabolism as a direct function of their concentrations. Qualitatively, therefore, alcohol dehydrogenase activity appears to be a major rate-limiting factor in ethanol metabolism. Quantitatively, however, the constants may not agree because of distribution in the animal or metabolism of the inhibitors. At saturating concentrations of inhibitors, ethanol is eliminated by inhibitor-insensitive pathways, at about 10% of the total rate at a dose of ethanol of 10 mmol/kg. Uncompetitive inhibitors of alcohol dehydrogenase should be especially useful for inhibiting the metabolism of alcohols since they are effective even at saturating levels of alcohol, in contrast to competitive inhibitors, whose action is overcome by saturation with alcohol.

Alcohol Dehydrogenase↗
PubMed1983

Involvement of histidine residues in the activity of horse liver alcohol dehydrogenase.

X-ray crystallographic studies indicate that His-51 in alcohol dehydrogenase may participate in a proton relay system during enzymatic catalysis [Eklund, H., Plapp, B. V., Samama, J.-P., & Brändén, C.-I. (1982) J. Biol. Chem. 257, 14349-14358], but there is no direct chemical evidence for this role. Diethyl pyrocarbonate (0.5-2 mM, pH 8, 25 degrees C) rapidly inactivated alcohol dehydrogenase, which was acetimidylated on all accessible lysine residues in order to prevent their modification. The reaction appeared to be specific for histidine residues, and the enzyme could be reactivated with 0.5 M hydroxylamine. The ethoxyformylated enzyme could still bind coenzymes, substrate analogues, and bipyridine, but with decreased affinity. The relationship between enzyme activity and the number of histidine residues modified showed that two histidine residues are modified during inactivation. NADH and isobutyramide significantly reduced the rate of inactivation, and the loss of activity then correlated with the modification of one to two histidine residues. The pH dependence of the inactivation showed the unusually high pK value of 9.6, which we attribute to the ionization of the water bound to zinc in the proton relay system. Although the histidine residue involved in the inactivation has not been identified, we conclude that one histidine residue (probably His-51) is essential for enzymatic activity.

Alcohol Dehydrogenase↗
PubMed1983

omega-haloalkyl esters of 5'-adenosine monophosphate as potential active-site-directed reagents for dehydrogenases.

A series of esters of adenosine 5'-monophosphate with ethyl, propyl, or hexyl moieties substituted at the omega-position with chlorine or bromine were prepared. The compounds were competitive inhibitors of horse liver alcohol dehydrogenase with respect to coenzyme, NAD+, and had inhibition (dissociation) constants in the range of 40 to 260 microM at pH 8.0, 25 degrees C. The bromoalkyl esters were designed to be active-site-directed inactivators and were chemically reactive as tested with the model compound 4-(p-nitrobenzyl)pyridine. Yeast alcohol dehydrogenase was inactivated by the bromohexyl analog by an active-site-directed mechanism, with a Ki = 1.5 mM and a pseudo-bimolecular rate constant of 0.03 M-1 S-1, which is 150 times larger than the bimolecular rate constant for inactivation by 2-bromoethanol. However, the rates of inactivation of other dehydrogenases treated with 10 mM concentrations of these compounds were generally slower than with the simpler reagent, 2-bromoethanol. Thus, the reactive functional group attached to the AMP moiety may not be properly oriented for affinity labeling of these dehydrogenases. The bromoalkyl esters may be useful for inactivating other enzymes.

Adenosine Monophosphate↗
PubMed1983

Inhibition by carboxamides and sulfoxides of liver alcohol dehydrogenase and ethanol metabolism.

Sulfoxides and amides were tested as inhibitors of liver alcohol dehydrogenase and ethanol metabolism in rats. With both series of compounds, increasing the hydrophobicity resulted in better inhibition, and introduction of polar groups reduced inhibition. Of the cyclic sulfoxides, tetramethylene sulfoxide was the best inhibitor as compared to the tri- and pentamethylene analogue and other compounds, and it may be a transition-state analogue. The most promising compounds, tetramethylene sulfoxide and isovaleramide, were essentially uncompetitive inhibitors of purified horse and rat liver alcohol dehydrogenases with respect to ethanol as substrate. These compounds also were uncompetitive inhibitors in vivo, which is advantageous since the inhibition is not overcome at higher concentrations of ethanol, as it is with competitive inhibitors, such as pyrazole. The uncompetitive inhibition constants for tetramethylene sulfoxide and isovaleramide for rat liver alcohol dehydrogenase were 200 and 20 microM, respectively, in vitro, whereas in vivo the values were 340 and 180 mumol/kg. The differences in the values may be due to metabolism or distribution of the compounds. Further studies will be required to determine if isovaleramide or tetramethylene sulfoxide is suitable for therapeutic purposes.

Alcohol Dehydrogenase↗
PubMed1982

Binding of substrate in a ternary complex of horse liver alcohol dehydrogenase.

Horse liver alcohol dehydrogenase was crystallized from an equilibrium mixture containing predominantly NAD+ and p-bromobenzyl alcohol. X-ray diffractometer data to a resolution of 2.9 A were collected and used to compute electron density maps with phases calculated from the isomorphous enzyme . NADH . dimethyl sulfoxide complex, which has been refined to an R value of 25.6%. The electron density maps were readily interpreted in a graphics display system. Both subunits of the dimer bind coenzyme and alcohol in essentially the same manner; there is no evidence of asymmetry between subunits. The bromophenyl group is accommodated in a large hydrophobic pocket that has the side chain of Leu-116 rotated into a different position than in the complex with dimethyl sulfoxide. The alcohol oxygen is directly ligated to the catalytic zinc atom. The zinc is tetracoordinate and there is no room for a water molecule to make the zinc pentacoordinate. A hydrogen-bonded system formed with the hydroxyl groups of the alcohol, Ser-48 and nicotinamide ribose (2'), and the imidazole of His-51 may provide a proton relay system that links the buried alcohol to solvent. The insertion of the coenzyme's hydroxyl group into this system appears to install the catalytically active species. The observed structure has the pro-R hydrogen on C1 of the alcohol pointing away from C4 of the nicotinamide ring. This is probably a nonproductive complex that easily becomes productive by a rapid rotation of the alcohol to put the pro-R hydrogen within 3 A of C4 of the nicotinamide ring and in position for a direct transfer of hydrogen. A model of the productive complex readily explains the stereospecificity of hydride transfer observed for ethanol.

Alcohol Dehydrogenase↗
PubMed1981

Inactivation of liver alcohol dehydrogenases and inhibition of ethanol metabolism by ambivalent active-site-directed reagents.

Active-site-directed reagents, of the general structure omega-(BrCH2CONH)RCOY, where R = alkyl, aryl, or aralkyl, and Y = OH or NH2, inactivated horse, mouse, rat, and human liver alcohol dehydrogenases at widely different rates, reflecting differences in reagent specificity and in the structures of the enzymes. Treatment of mice and rats with either of two optimally specific reagents, p-(XCH2CONH)C6H4(CH2)3CONH2, where X = Br (7) or CH3SO3 (10), partially (20 to 40%) inactivated alcohol dehydrogenase in liver, inhibited ethanol metabolism, and prolonged the impairment of coordination produced by ethanol in these animals. Although the dose of 7 used (0.13 mmol/kg) approximated the LD50, 10 was effective at a dose of 0.48 mmol/kg that was not acutely toxic.

Alcohol Oxidoreductases↗
PubMed1981

Sulfonate analogues of adenosine nucleotides as inhibitors of nucleotide-binding enzymes.

2-(Adenin-9-yl)ethanesulfonic acid (1), 3-(adenin-9-yl)propanesulfonic acid (2), 9-(5-deoxy-beta-D-ribofuranosyl)-adenine-5'-sulfonic acid (3), and 9-(3-deoxy-beta-D-arabinofuranosyl)adenine-3'-sulfonic acid (4) were prepared by reaction of the corresponding chlorides by sodium sulfite (1-3) or by reaction of an epoxide with sodium hydrogen sulfite (4). They inhibited a typical nucleotide-binding enzyme, horse liver alcohol dehydrogenase, with inhibition constants in the range of 0.18-4.9 mM at pH 8, 25 degrees C.

Adenine Nucleotides↗
PubMed1980

In vivo evaluation of ambivalent active-site-directed inactivators of liver alcohol dehydrogenase.

In order to decrease the rate of ethanol metabolism for the treatment of acute and chronic alcoholism it would be useful to inhibit liver alcohol dehydrogenase in vivo. Based on a knowledge of the three-dimensional structure of the horse enzyme, we designed active-site-directed inactivators [p-(XCH2CONH)C6H4(CH2)3COHN2] which bind to the enzyme-NAD or enzyme-NADH complex and alkylate methionine residue 306. In vitro, these reagents inactivated mouse, rat, horse and human liver alcohol dehydrogenases faster in the presence than in the absence of NAD or NADH, but with slightly different specificity. Mice and rats pretreated with the reagents eliminated ethanol in blood more slowly than those not treated, and the specific activity of alcohol dehydrogenase in liver homogenates of treated animals was decreased. It appears that the design of active-site-directed reagents is feasible, but these reagents must be improved so that they are more efficacious in vivo.

Alcohol Oxidoreductases↗