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W F Bosron

Publications and source records attributed to W F Bosron.

At least 37 records · Page 2Linked to original sources
PubMed1995

Cimetidine inhibition of human gastric and liver alcohol dehydrogenase isoenzymes: identification of inhibitor complexes by kinetics and molecular modeling.

Cimetidine, an H2-receptor antagonist, is one of the most commonly prescribed drugs in the world. It has been reported to increase blood alcohol concentrations in drinking individuals. To determine if this increase could be due to inhibition of alcohol dehydrogenase activity, the effect of the drug on ethanol oxidation by gastric sigma sigma alcohol dehydrogenase and liver beta 2 beta 2, pi pi, and chi chi alcohol dehydrogenase isoenzymes was observed. Cimetidine inhibited all isoenzymes studied except chi chi; the chi chi isoenzyme showed no inhibition up to 5 mM cimetidine. Inhibition of the alcohol dehydrogenase isoenzymes by the H2-receptor antagonists nizatidine, ranitidine, and famotidine was negligible. Docking simulations with the beta 2.NAD+.4-iodopyrazole X-ray structure indicated that cimetidine fit well into the substrate binding site. The substitution on the thiazole ring of nizatidine, however, prevented docking into the binding site. Cimetidine inhibition of ethanol oxidation by sigma sigma and beta 2 beta 2 was competitive with varied ethanol, exhibiting Ki values of 2.8 +/- 0.4 mM and 0.77 +/- 0.07 mM, respectively. Cimetidine inhibition of ethanol oxidation by pi pi was noncompetitive with varied ethanol (Ki = 0.50 +/- 0.03 mM). Inhibition of ethanol oxidation by sigma sigma and beta 2 beta 2 with varied NAD+ was competitive. These results, together with the cimetidine inhibition kinetics of acetaldehyde reduction by sigma sigma and beta 2 beta 2, with either varied NADH or varied acetaldehyde, are consistent with cimetidine binding to two enzyme species. These species are free enzyme and the productive enzyme.NAD+ complex.

Alcohol Dehydrogenase↗
PubMed1995

Expression and kinetic characterization of recombinant human stomach alcohol dehydrogenase. Active-site amino acid sequence explains substrate specificity compared with liver isozymes.

A full-length 1966-base pair clone of the human class IV alcohol dehydrogenase (sigma-ADH) was isolated from a human stomach cDNA library. The 373-amino acid sigma-ADH encoded by this cDNA was expressed in Escherichia coli. The specific activity of the recombinant enzyme for ethanol oxidation at pH 7.5 and 25 degrees C, calculated from active-site titration of NADH binding, was 92 +/- 9 units/mg. Kinetic analysis of the catalytic efficiency (kcat/KM) of recombinant sigma-ADH for oxidation of primary alcohols indicated broad substrate specificity. Recombinant human sigma-ADH exhibited high catalytic efficiency for oxidation of all-trans-retinol to all-trans-retinal. This pathway is important in the synthesis of the transcriptional regulator all-trans-retinoic acid. Secondary alcohols and 3 beta-hydroxysteroids were inactive with sigma-ADH or were oxidized with very low efficiency. The KM of sigma-ADH for ethanol was 25 mM, and the KM for primary straight chain alcohols decreased substantially as chain length increased. There are important amino acid differences in the alcohol-binding site between the human class IV (sigma) and human class I (beta) alcohol dehydrogenases that appear to explain the high catalytic efficiency for all-trans-retinol, the high kcat for ethanol, and the low catalytic efficiency for secondary alcohols of sigma-ADH relative to beta 1-ADH. For example, modeling the binding of all-trans-retinol in the human beta 1-ADH structure suggested that coordination of retinol to the active-site zinc is hindered by a loop from residues 114 to 120 that is at the entrance to the alcohol-binding site. The deletion of Gly-117 in human sigma-ADH and a substitution of Leu for the bulky Tyr-110 appear to facilitate retinol access to the active-site zinc.

Alcohol Dehydrogenase↗
PubMed1995

Tissue distribution of cocaine methyl esterase and ethyl transferase activities: correlation with carboxylesterase protein.

The tissue distribution of cocaine methyl esterase and ethanol-dependent ethyl transferase activities was determined in the rat and compared to the tissue distribution of three distinct non-specific hydrolases. Rates of formation of benzoylecgonine from cocaine and cocaethylene from ethanol and cocaine were measured in serum and tissue homogenate-supernatants of the brain, heart, kidney, liver, lung and spleen. The tissue distribution of three nonspecific esterases, A, B and C, was defined by nondenaturing gel electrophoresis and measuring the hydrolysis of 4-methylumbelliferyl acetate in the gels. Immunoreactive protein was localized by using Western blot analysis with polyclonal rabbit antihuman liver cocaine methyl esterase antibody after denaturing and nondenaturing gel electrophoresis. The rat liver, lung, kidney and heart exhibited cocaine methyl esterase and ethyl transferase activities and immunoreactive protein. The brain had cocaine methyl esterase activity but no ethyl transferase activity; neither activity was found in serum or spleen. The dominant immunoreactive bands in the liver, lung, kidney and heart comigrated with the 59 kD band of purified human liver cocaine methyl esterase. The rat liver, lung and kidney exhibited a band of nonspecific esterase activity that migrated with purified human liver cocaine methyl esterase and rat hydrolase A. These observations suggest that rat hydrolase A is similar to human cocaine methyl esterase. The lack of straight forward correlation between cocaine methyl esterase activity and immunoreactive protein and nonspecific esterase activity suggests that more than one enzyme catalyzes the hydrolysis of cocaine to benzoylecgonine in the rat.

Animals↗
PubMed1994

Purification and characterization of a human liver cocaine carboxylesterase that catalyzes the production of benzoylecgonine and the formation of cocaethylene from alcohol and cocaine.

The psychomotor stimulant cocaine is inactivated primarily by hydrolysis to benzoylecgonine, the major urinary metabolite of the drug. A non-specific carboxylesterase was purified from human liver that catalyzes the hydrolysis of the methyl ester group of cocaine to form benzoylecgonine. In the presence of ethanol, the enzyme also catalyzes the transesterification of cocaine producing the pharmacologically active metabolite cocaethylene (benzoylecgonine ethyl ester). The carboxylesterase obeys simple Michaelis-Menten kinetics with Km values of 116 microM for cocaine and 43 mM for ethanol. The enzymatic activity suggests that it may play an important role in regulating the detoxication of cocaine and in the formation of the active metabolite cocaethylene. Additionally, the enzyme catalyzes the formation of ethyloleate from oleic acid and ethanol. The carboxylesterase was purified from autopsy liver by gel filtration, chromatofocusing, ion-exchange, and hydrophobic interaction chromatography to purity by SDS-PAGE and agarose gel isoelectric focusing. The subunit molecular weight was determined to be 59,000 and the native molecular weight was estimated to be 170,000 from a calibrated gel filtration column, suggesting that the active enzyme is a trimer. The isoelectric point was approximately 5.8. Digestion of carbohydrate residues on the protein with an acetylglucosaminidase plus binding to several lectins indicates that the enzyme is glycosylated. The esterase was cleaved with two proteases, and the amino acid sequences from fourteen peptides were used to search GenBank. Two identical matches were found corresponding to carboxylesterase cDNAs from human liver and lung.

Amino Acid Sequence↗
PubMed1994

Structures of three human beta alcohol dehydrogenase variants. Correlations with their functional differences.

The three-dimensional structures of three variants of human beta alcohol dehydrogenase have been determined to 2.5 A resolution. These three structures differ only in the amino acid at position 47 and the molecules occupying the alcohol binding site. Human beta 1 alcohol dehydrogenase has an Arg at position 47 and was crystallized in a complex with NAD(H) and cyclohexanol. A naturally occurring variant of beta 1 alcohol dehydrogenase, found in approximately 50% of the Asian population, possesses a His at position 47 (beta 2 or beta 47H) and was crystallized in a complex with NAD+ and the inhibitor 4-iodopyrazole. A site-directed mutant of beta 1 alcohol dehydrogenase in which a Gly is substituted for Arg47 (beta 47G) was crystallized in a complex with NAD+. By comparing both the common and unique features of these structures, it is clear that position 47 contributes significantly to the strength of protein-coenzyme interactions. The substitution of Arg47 by His produces an enzyme with a 100-fold lower affinity for coenzyme, but creates no large changes in the enzyme structure. The substitution of Arg47 by Gly produces an enzyme with coenzyme binding characteristics more similar to the wild-type enzyme than to the enzyme with His at position 47, but the structure of the Gly47 variant exhibits differences in and around the coenzyme binding site. These changes involve a rigid-body rotation of the catalytic domain towards the coenzyme domain by approximately 0.8 degrees and local rearrangements of amino acid side-chains, such as a 1.0 A movement of Lys228, relative to the beta 1 enzyme. These structural alterations may compensate for the loss of coenzyme interactions contributed by Arg47 and can explain the high affinity of the Gly47 variant for coenzyme.

Alcohol Dehydrogenase↗
PubMed1994

Comparative roles of histidine 51 in human beta 1 beta 1 and threonine 51 in pi pi alcohol dehydrogenases.

Histidine at position 51 of the class I beta 1 alcohol dehydrogenase (ADH) functions as a general base by indirectly abstracting a proton from the alcohol substrate through a hydrogen-bonded proton relay system. The human class II pi-ADH was reported to be polymorphic, having either Ser or Thr, but not His at position 51. It is unknown whether Ser or Thr51 have a catalytic role in ethanol oxidation with pi-ADH. Accordingly, we expressed and purified recombinant mutants of pi-ADH with Thr, Ser, and His at position 51. At pH 6.5, values for Vmax/Km for ethanol were 0.30, 0.10, and 0.09 min-1 mM-1 for pi 51Thr, pi 51Ser, and pi 51His ADH, respectively. Hence the effects of the substitutions were much less than the 11-fold decrease in Vmax/Km observed for beta 1-ADH when a neutral amino acid (Gln) was substituted for His51. Addition of a buffer base (400 mM glycylglycine) had little effect on Vmax/Km of recombinant pi 51Thr or pi 51Ser ADH, while it increased Vmax/Km for ethanol 7-fold for the beta (1)51 Gln ADH. We conclude that there is no evidence for Thr51 of pi-ADH participating in a proton relay similar to that seen in beta 1-ADH and that a base at position 51 may not be a universal requirement for a functional alcohol dehydrogenase with a moderate efficiency for ethanol oxidation at a physiological pH.

Alcohol Dehydrogenase↗
PubMed1994

Catalytic efficiency of human alcohol dehydrogenases for retinol oxidation and retinal reduction.

Mammalian alcohol dehydrogenase (ADH) is thought to be involved in the reversible oxidation of vitamin A or retinol to retinal for retinoic acid synthesis. Retinoic acid is a potent transcriptional regulator and a morphogen. It was proposed that the competition of consumed ethanol with retinol oxidation by ADH might explain developmental disorders seen with fetal alcohol syndrome. We report herein the relative efficiency (V/Km) of eight human ADH isoenzymes for oxidation of all-trans-retinol and reduction of three retinal isomers (all-trans, 9-cis, and 13-cis-retinal). Class IV sigma sigma and class II pi pi isoenzymes are the most efficient forms, with V/Km values approximately 100 and 30 times greater, respectively, than class I beta 1 beta 1 or gamma 1 gamma 1, sigma sigma exhibits the highest V/Km (1-2 microns-1min-1), followed by pi pi, with V/Km of 0.5-0.6 microns-1min-1 for all-trans-retinol, all-trans-retinal, and 9-cis-retinal. pi pi also has the lowest Km (11-14 microns) for all-trans-retinol and three retinal isomers. alpha alpha shows an intermediate efficiency, with V/Km of 0.09-0.2 microns-1min-1 and a relatively low Km of 16-24 microns for all four substrates. alpha alpha has the highest efficiency of all tested isoenzymes for 13-cis-retinal. Class III chi chi is inactive with all the tested retinoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1993

Monomers of human beta 1 beta 1 alcohol dehydrogenase exhibit activity that differs from the dimer.

A previously unreported enzymatic activity is described for monomers of the beta 1 beta 1 isoenzyme of human alcohol dehydrogenase that were prepared from dimeric enzyme by freeze-thaw in liquid nitrogen. Whereas the dimeric enzyme has optimal activity at low substrate concentrations (2.5 mM ethanol, 50 microM NAD+; "low Km" activity), the monomer has its highest activity at high substrate concentrations (1.5 M ethanol, 2.5 mM NAD+; "high Km" activity). While the activity of the monomer does not appear to be saturated at 1.5 M ethanol, its maximal activity at this high ethanol concentration exceeds the Vmax of the dimer by about 3-fold. The apparent Km of NAD+ with monomers is 270 microM, and no activity could be detected with nicotinamide mononucleotide as cofactor. During gel filtration the high Km activity elutes at a lower apparent molecular weight position than the dimer. The kinetics of monomer-to-dimer reassociation are consistent with a second-order process with a rate constant of 240 M-1 s-1. The reassociation rate is markedly enhanced by the presence of NAD+. During refolding of beta 1 beta 1 following denaturation in 6 M guanidine hydrochloride, an enzyme species with high Km activity and spectral properties similar to the freeze-thaw monomer is observed, indicating that a catalytically active monomer is an intermediate in the refolding pathway. The enzymatic activity of the monomer implies that the intersubunit contacts of beta 1 beta 1 are not crucial in establishing a catalytically competent enzyme. However, the differences in specific activity and Km between monomer and dimer suggest that dimerization may serve to modulate the catalytic properties.

Alcohol Dehydrogenase↗
PubMed1993

Amino acid substitutions at position 47 of human beta 1 beta 1 and beta 2 beta 2 alcohol dehydrogenases affect hydride transfer and coenzyme dissociation rate constants.

Human liver alcohol dehydrogenase isoenzymes beta 1 beta 1 and beta 2 beta 2, in which position 47 in the coenzyme binding domain is an arginine or histidine, respectively, differ remarkably in steady-state kinetics. To understand which catalytic steps affect these kinetics, apparent coenzyme dissociation and association rate constants, and apparent 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) hydride transfer rate constants were obtained with stopped-flow kinetics. Enzymes containing site-specific mutations of Arg-47 in beta 1 beta 1 (beta 47R) to His (beta 2 beta 2 or beta 47H), Lys (beta 47K), or Gln (beta 47Q) were studied. Apparent coenzyme dissociation rate constants are greatly affected by substitutions at position 47, in which mutant enzymes with a weak base or a neutral residue at this position (beta 47H and beta 47Q) exhibit faster rate constants than beta 47R and beta 47K. Substitutions at position 47 have less effect on apparent coenzyme association rate constants. The kinetics of NADH association for beta 47H and beta 47Q are consistent with a two-step mechanism in which the bimolecular binding step is coupled to a unimolecular process. These findings indicate that the greater role of position 47 in coenzyme dissociation may occur after a coenzyme-induced isomerization. Substitutions at position 47 also strongly influence apparent DACA hydride transfer rate constants; hydride transfer is faster with mutant enzymes containing weak bases like histidine at this position. Steady-state kinetics, however, reveal that the rate-limiting step of both beta 47R and beta 47H for acetaldehyde reduction and for ethanol oxidation is coenzyme product dissociation. Thus, the different activities of beta 1 beta 1 and beta 2 beta 2 for ethanol oxidation and acetaldehyde reduction are caused primarily by different coenzyme dissociation rates.

Alcohol Dehydrogenase↗
PubMed1993

Purification and partial amino acid sequence of a high-activity human stomach alcohol dehydrogenase.

To understand the relative importance of alcohol dehydrogenase (ADH) isoenzymes in gastric ethanol metabolism, a stomach-specific ADH (sigma-ADH) was purified to homogeneity from human transplant donor and surgical tissues, and its activity for ethanol oxidation was examined. The enzyme from these tissues had a specific activity at pH 10 of approximately 70 units/mg, about 10 times that reported by Moreno and Parés (J. Biol. Chem. 266:1128-1133, 1991). The enzyme exhibited a high Km for ethanol at pH 7.5 and 10 (29 and 5.2 mM, respectively). This high-activity sigma-ADH isoenzyme migrated on starch and isoelectric focusing gels to a position slightly anodic to the liver pi pi isoenzyme. It was subjected to digestion by endoproteinases, and approximately 40% of the protein was sequenced. The sigma-ADH exhibited 75%, 68%, and 62% sequence identity to the human class I (beta 1), II (pi), and III (chi) isoenzymes, respectively, and 61% identity to the deduced ADH6 amino acid sequence. Phylogenetic analysis indicated that precursors to this high-activity sigma-ADH and the class I isoenzymes diverged more recently than precursors to the class II and III isoenzymes, after reptilian and avian divergence. The high-activity sigma-ADH isoenzyme therefore represents a distinct class of ADH (class IV), more closely related in evolution to the class I isoenzymes than to the other known human isoenzymes.

Adult↗
PubMed1992

Tryptophan fluorescence quenching by alkaline pH and ternary complex formation in human beta 1 beta 1 and horse EE alcohol dehydrogenases.

The horse EE and human beta 1 beta 1 alcohol dehydrogenase isoenzymes have almost identical protein backbone folding patterns and contain 2 tryptophans per subunit (Trp-15 and Trp-314). Tyr-286, which had been proposed to quench the fluorescence of Trp-314 by resonance energy transfer at alkaline pH in EE, is substituted by Cys in beta 1 beta 1. The proposed role of Tyr-286 in pH-dependent quenching of EE is confirmed by our observation that tryptophan fluorescence of beta 1 beta 1 is not substantially quenched at alkaline pH. Tyr-286 had also been implicated in the quenching of Trp-314 upon formation of the EE-NAD(+)-trifluoroethanol ternary complex. However, beta 1 beta 1 exhibits the same extent of tryptophan fluorescence quenching as EE upon complexation, which strongly suggests that Tyr-286 is not involved in ternary complex quenching.

Alcohol Dehydrogenase↗
PubMed1992

Human alcohol dehydrogenase: dependence of secondary alcohol oxidation on the amino acids at positions 93 and 94.

The human liver alpha alpha and beta 1 beta 1 isoenzymes are straight-chain alcohol dehydrogenases with different efficiencies toward secondary alcohols. Two of the 24 amino acid substitutions in alpha alpha (A for F93 and I for T94) were made by site-directed mutagenesis of beta 1 beta 1 and the substrate specificity of beta 93A94I was examined. The Vmax/KM values of beta 93A94I for secondary alcohols (especially R enantiomers) are similar to that of alpha alpha and as much as 4000-fold greater than beta 1 beta 1, but the dependences of Vmax/KM on primary alcohol chain length are similar to beta 1 beta 1, but not alpha alpha. Thus, the substitutions of A for F93 and I for T94 in beta 1 beta 1 account for the increased efficiency towards secondary alcohols and stereoselectivity for enantiomeric alcohols, but not for the effects of chain length on the Vmax/KM for primary alcohols seen with alpha alpha.

Alcohol Dehydrogenase↗
PubMed1992

Effects of ethanol on cocaine metabolism: formation of cocaethylene and norcocaethylene.

The coabuse of cocaine and ethanol occurs with high frequency and increases the risk of cocaine-related morbidity and mortality. The mechanisms mediating the toxic interactions of cocaine and ethanol are not clearly defined. This study examined the effects of acute ethanol administration on the metabolism of cocaine in the male Wistar rat. Intraperitoneal administration of 2 g/kg ethanol 30 min prior to administration of 25 mg/kg cocaine resulted in the formation of two ethylated derivatives of cocaine, benzoylecgonine ethyl ester (cocaethylene) and benzoylnorecgonine ethyl ester (norcocaethylene) in liver, brain, and serum. Fifteen minutes after cocaine administration, the tissue levels of cocaethylene were 22, 10, and 9% of the cocaine recovered from liver, serum, and brain, respectively. Ethanol pretreatment increased cocaine concentrations in liver and benzoylnorecgonine concentrations in liver and serum. The increased morbidity and hepatotoxicity seen with acute combined administration of cocaine and ethanol may be due to the formation of the toxic ethylated and N-demethylated metabolites of cocaine. Ethanol pretreatment decreased benzoylecgonine concentrations in serum and liver. The most important consequence of ethanol-induced inhibition of the normally rapid hydrolysis of cocaine to benzoylecgonine may be a decrease in benzoylecgonine-mediated vasoconstriction.

Animals↗
PubMed1991

Structure of human beta 1 beta 1 alcohol dehydrogenase: catalytic effects of non-active-site substitutions.

The three-dimensional structure of human beta 1 beta 1 alcohol dehydrogenase (ADH; EC 1.1.1.1) complexed with NAD+ has been determined by x-ray crystallography to 3.0-A resolution. The amino acids directly involved in coenzyme binding are conserved between horse EE and human beta 1 beta 1 alcohol dehydrogenase in all but one case [serine (horse) vs. threonine (human) at position 48]. As a result, the coenzyme molecule is bound in a similar manner in the two enzymes. However, the strength of the interactions in the vicinity of the pyrophosphate bridge of NAD+ appears to be enhanced in the human enzyme. Side-chain movements of Arg-47 and Asp-50 and a shift in the position of the helix comprising residues 202-212 may explain both the decreased Vmax and the decreased rate of NADH dissociation observed in the human enzyme vs. the horse enzyme. It appears that these catalytic differences are not due to substitutions of any amino acids directly involved in coenzyme binding but are the result of structural rearrangements resulting from multiple sequence differences between the two enzymes.

Alcohol Dehydrogenase↗