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B V Plapp

Publications and source records attributed to B V Plapp.

At least 73 records · Page 4Linked to original sources
PubMed1980

Kinetics and control of alcohol oxidation in rats.

The rates of oxidation of ethanol and isopropanol by purified rat liver alcohol dehydrogenase were determined in vitro and compared to the rates of metabolism in vivo in order to estimate the extent to which alcohol dehydrogenase activity limits ethanol metabolism. The metabolism of isopropanol and isopropanol-d7 (CD3CDOHCD3) was examined by measuring blood alcohol and acetone levels at various times and apparently proceeds by an irreversible, enzyme-catalyzed pathway: isopropanol leads to acetone leads to an unidentified metabolite. The kinetic constants for the metabolism were computed from simultaneous fits to the appropriate differential equations using a nonlinear least-squares program. The relative rates of oxidation of the alcohols, ethanol:isopropanol:isopropanol-d7, at 25 mM were 9.6 : 2.3 : 1.0 in vitro and 4.1 : 2.4 : 1.0 in vivo. Since the ratio of rates for isopropanol is about the same in vitro and in vivo, it appears that alcohol dehydrogenase activity is the predominant rate-limiting factor in isopropanol metabolism. The relatively slower rate of ethanol oxidation in vivo as compared to in vitro suggests that liver alcohol dehydrogenase is partially (about 40%) limiting for ethanol metabolism.

1-Propanol↗
PubMed1979

Kinetics of carboxymethylation of histidine hydantoin.

The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.

Acetates↗
PubMed1979

3-Substituted pyrazole derivatives as inhibitors and inactivators of liver alcohol dehydrogenase.

3-Substituted pyrazoles, HOCH2 (1), HOCH2CH2 (2), HOCH2CH2CH2 (3), ClCH2 (4), ClCH2CH2 (5), ClCH2CH2CH2 (6), and CH3CO (7), were synthesized and evaluated in vitro on horse liver alcohol dehydrogenase for their potential as inhibitors of ethanol metabolism. 1 to 6 bound to the enzyme-NAD+ complex with dissociation constants of 40 to 200 microM, much higher than the constants for the corresponding 4-substituted pyrazoles, but with the same absorption maximum at 295 nm. 4 inactivated the enzyme within a few minutes, but NAD+ protected against reaction, and 4 nonspecifically alkylated many sulfur atoms in the protein. The isomer, 4-(chloromethyl)pyrazole, behaved similarly, 5 and 6 strongly inhibited the enzyme in the presence of NAD+, due to formation of the slowly dissociable (10(-3)s-1) enzyme-NAD+-pyrazole complex, but did not irreversibly inactivate the enzyme. 7 inhibits the enzyme weakly (Kp = 5 mM). It appears that the 3-substituted pyrazoles bind to the enzyme-NAD+ complex with the reactive functional group improperly positioned for specific irreversible reaction.

Alcohol Oxidoreductases↗
PubMed1978

pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases.

Horse liver alcohol dehydrogenase specifically carboxymethylated on cysteine-46 (a ligand to the zinc in the active site) or acetimidylated on 25 of the 30 lysine residues per subunit (including residue 228) was studied. The tryptophan fluorescence of these enzymes decreased by 35% as pH was increased, with an apparent pKa of 9.8 +/- 0.2, identical with that of native enzyme. Native enzyme in the presence of 30mM-imidazole, which displaces a water molecule ligated to the zinc, also had a pKa of 9.8. The ionoizable group is thus neither the water molecule nor one of the modified groups. Binding of NAD+ shifted the pKa for the fluorescence transition to 7.6 with native enzyme and to 9.0 with acetimidylated enzyme, but did not shift the pKa of carboxymethylated enzyme. Binding of NAD+ and trifluoroethanol, an unreactive alcohol, gave maximal fluorescence quenching at pH7 with all three enzymes. The acetimidylated enzyme--NAD+--trifluoroethanol complex had an apparent pKa of 5.0, but the pK of the native enzyme complex was experimentally inaccessible. The results are interpreted in terms of coupled equilibria between two different conformational states. On binding of NAD+, the modified enzymes apparently change conformation less readily than does native enzyme, but binding of alcohol can drive the change to completion.

Alcohol Oxidoreductases↗
PubMed1977

Characterization and kinetics of native and chemically acitvated human liver alcohol dehydrogenases.

Acetimidylation of the amino groups of alcohol dehydrogenase from human and horse liver yields several modified enzyme forms, which differ in electrophoretic mobility and can be separated by ion exchange chromatography, but which are similar in kinetic characteristics. The acetimidylated, as well as the methylated, enzymes from human livers of the normal phenotype have increased activity and larger Michaelis and inhibition constants. These results suggest that the human enzyme has amino groups at the active sites, as was shown previously for the horse enzyme. The variant subunit occuring in the enzyme isolated from atypical human livers does not seem to be activated by acetimidylation, which may indicate that substitution of proline for Ala-230 or modifiction of Lys-228 is sufficient to fully activate the enzyme. Results of product inhibition studies of native and modified human enzymes are consistent with an Ordered Bi Bi mechanism. However, the major isoenzyme of native human liver alcohol, dehydrogenase exhibits nonlinear kinetics over a wide range of ethanol concentrations. This result may indicate that subunits with different kinetic characteristics are present or that there is negative cooperativity between subunits. After chemical modification, the kinetic patterns become linear, suggesting that the mechanism is altered.

Acetaldehyde↗
PubMed1977

Kinetics of native and activated isozymes of horse liver alcohol dehydrogenase.

The major isozymes of horse liver alcohol dehydrogenase (EC 1.1.1.1), EE, ES, and SS, have been separated by chromatography on phosphocellulose. Product inhibition studies showed that the kinetic behavior of EE and SS isozymes was consistent with the ordered BiBi mechanism. The different primary structures of the E and S subunits were expressed with higher Michaelis constants for ethanol and acetaldehyde and lower activity for the SS isozyme when compared with the EE isozyme. The differences for SS isozyme are reflections of slower rates of association and dissociation of coenzymes and slower rates of hydrogen transfer, not of affinities for the substrates. The contributions of each subunit in ES isozyme to the kinetic constants were not additive, indicating that the subunits may not act independently. Activation of the isozymes by amidination and alkylation suggested that lysine residues were present at the active sites of both E and S subunits. Kinetic studies indicated that isonicotinimidylation increased enzyme activity of the three isozymes by increasing the rates of dissociation of the enzyme-coenzyme complexes.

Alcohol Oxidoreductases↗
PubMed1976

Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with diazonium-1H-tetrazole.

Diazonium-1H-tetrazole was tested as a potential active-site-directed reagent for amino acid residues involved in catalysis by alcohol dehydrogenase. In a novel reaction with a protein, diazonium-1H-tetrazole inactivated the enzyme selectively, and almost stoichiometrically, but reacting with the sulfur of a cysteine residue, Cys-174. As a model compound, the tetrazole adduct of free cysteine was prepared. Elementary and spectral analyses of the adduct were consistent with the structure 5-tetrazoleazo-S-cysteine. The adduct absorbs light with a maximun at 316 nm, and is destroyed by irradiation at this wavelength. The inactivated enzyme still bound NADH as determined by difference spectroscopy, but did not enhance the fluorescence of the bound NADH as did native enzyme. X-ray crystallographic studies of free enzyme have shown that Cys-174 coordinates the zinc at the active site (Eklund, H., Nordström, B., Zeppezauer, E., Söderlund, G., Ohlsson, I., Boiwe, T., and Brändén, C-I. (1974), FEBS Lett. 44, 200-204). The modified enzyme is probably inactive because the large, negatively charged tetrazole ring interferes sterically or electrostatically with the binding of substrates or with hydride transfer.

Alcohol Oxidoreductases↗
PubMed1975

Activation of liver alcohol dehydrogenases by imidoesters generated in solution.

Various omega-halogenated carboxy acids and amides were evaluated as potential active-site-directed reagents for alcohol dehydrogenase. 2-Bromoacetamide and bromoacetic and 3-bromopropionic acids inactivated the enzyme; AMP, NAD+, and NADH markedly decreased the rate of inactivation. Some omega-halogenated carboxyamides, X(CH2)nCONH2, increased the activity of the enzyme with the rate and extent of activation depending on the number of methylene units (n) in the order 3 greater than 4 greater than 2 and on X in the order Br greater than Cl. 4-Chlorobutyramide (0.1 M) activated the horse liver enzyme 20-fold in 24 hr at pH 8.0 and 25 degrees. The activation was not prevented by AMP or 2,2-bipyridine, but was by NADH. The kinetic constants and turnover numbers for human and horse liver alcohol dehydrogenases treated with chlorobutyramide were increased markedly compared to those for native enzymes. Alcohol dehydrogenase treated with chlorobutyramide was not further activated by methyl picolinimidate, an imidoester which activates native enzyme by modifying amino groups in the active sites. Chlorobutyramide does not appear to react directly with the enzyme but cyclizes in the reaction medium to form an intermediate imidoester, 2-iminotetrahydrofuran, which reacts with most of the amino groups of the enzyme.

Adenosine Monophosphate↗
PubMed1975

Identification of the lysine residue modified during the activation of acetimidylation of horse liver alcohol dehydrogenase.

A single amino group in horse liver alcohol dehydrogenase was modified with methyl(14C)acetimidate by a differential labeling procedure. Lysine residues outside the active site were modified with ethyl acetimidate while a lysine residue in the active site was protected by the formation of an enzyme-NAD+-pyrazole complex. After the protecting reagents were removed, the enzyme was treated with methyl(14C)acetimidate. Enzyme activity was enhanced 13-fold as 1.1 (14C)acetimidyl group was incorporated per active site. A labeled peptide was isolated from a tryptic-chymotryptic digest of the modified enzyme in 35% overall yield. Amino acid composition and sequential Edman degradations identified the peptide as residues 219-229; lysine residue 228 was modified with the radioactive acetimidyl group.

Acetates↗
PubMed1975

Activation and inactivation of horse liver alcohol dehydrogenase with pyridoxal compounds.

Pyridoxal compounds can either activate or inactivate horse liver alcohol dehydrogenase in differential labeling experiments. Amino groups outside of the active sites were modified with ethyl acetimidate, while the amino groups in the active sites were protected by the formation of the complex with NAD-plus and pyrazole. After removal of the NAD-plus and pyranzole, the partially acetimidylated enzyme was reductively alkylated with pyridoxal and NaBH4, with the incorporation of one pyridoxal group per subunit of the enzyme. The turnover numbers for the reaction of NAD-plus and ethanol increased by 15-fold, and for NADH and acetaldehyde by 32-fold. The Michaelis and inhibition constants increased 80-fold or more. Pyridoxal phosphate and NaBH4 also modified one group per subunit, but the turnover numbers decreased by 10-fold and the kinetic constants were intermediate between those obtained for pyridoxyl alcohol dehydrogenase and the partially acetimidylated enzyme. With native enzyme, the rates of dissociation of the enzyme-coenzyme complexes are rate-limiting in the catalytic reactions. The pyridoxyl enzyme is activated because the rates of dissociation of the enzyme-coenzyme complexes are increased. The rates of binding of coenzyme to phosphopyridoxyl enzyme have decreased due to the introduction of the negatively charged phosphate. The size of the group is not responsible for this decrease since these rates are not greatly decreased by the incorporation of pyridoxal. For both pyrodoxal and phosphopyridoxyl alcohol dehydrogenases, the interconversion of the ternary complex is at least partially rate-limiting. Chymotryptic-tryptic digestion of pryidoxyl enzyme produced a major peptide corresponding to residues 219 to 229, in which Lys 228 had reacted with pyridoxal. The same lysine residue reacted with pyridoxal phosphate.

Alcohol Oxidoreductases↗
PubMed1975

Rate-limiting steps in ethanol metabolism and approaches to changing these rates biochemically.

Ethanol is oxidized to acetate primarily by a system involving liver alcohol and aldehyde dehydrogenases coupled with reoxidation of NADH by the mitochondria. All of these steps are at least partially rate-limiting in ethanol metabolism, with alcohol dehydrogenase and oxidative phosphorylation probably slower than the others. More research is required to assess the quantitative roles of various steps. Many agents are ineffective in changing the rate of metabolism of ethanol, but fructose and dinitrophenol may increase the rate by up to 1.5-fold in vivo. The failure of single agents to increase the rate substantially may indicate that when one step is accelerated, another step becomes rate-limited. Therefore, combinations of agents that affect several steps simultaneously may be required for acceleration. Effective experimental methods for inhibiting alcohol dehydrogenase in vivo are available.

Acetaldehyde↗