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W F Bosron

Publications and source records attributed to W F Bosron.

At least 55 records · Page 3Linked to original sources
PubMed1991

General base catalysis in a glutamine for histidine mutant at position 51 of human liver alcohol dehydrogenase.

On the basis of the three-dimensional structure of horse liver alcohol dehydrogenase determined by X-ray crystallography, His 51 has been proposed to act as a general base during catalysis by abstracting a proton from the alcohol substrate. A hydrogen-bonding system (proton relay system) connecting the alcohol substrate and His 51 has been proposed to mediate proton transfer. We have mutated His 51 to Gln in the homologous human liver beta 1 beta 1 alcohol dehydrogenase isoenzyme which is expected to have a similar proton relay system. The mutation resulted in an about 6-fold drop in V/Kb (Vmax for ethanol oxidation divided by Km for ethanol) at pH 7.0 and a 12-fold drop at pH 6.5. V/Kb could be restored completely or partially by the presence of high concentrations of glycylglycine, glycine, and phosphate buffers. A Brønsted plot of the effect on V/Kb versus the pKa of these bases plus H2O and OH- was linear. Only secondary or tertiary amine buffers differed from linearity, presumably due to steric hindrance. These results suggest that His 51 acts as a general base catalyst during alcohol oxidation in the wild-type enzyme and can be functionally replaced in the mutant enzyme by general base catalysts present in the solvent. Steady-state kinetic constants for NAD+ and the trifluoroethanol inhibition patterns were similar between the wild-type and the mutant enzyme. Differences in the inhibition constants (Ki) of caprate and trifluoroethanol below pH 7.8 and in the pH dependence of Ki can be explained by the substitution of neutral Gln for positively charged His.

Alcohol Dehydrogenase↗
PubMed1991

Human liver cocaine esterases: ethanol-mediated formation of ethylcocaine.

A new, pharmacologically active metabolite of cocaine, ethylcocaine, has been reported in individuals after concurrent use of cocaine and ethanol. Formation of ethylcocaine may contribute to the common coabuse of these two drugs and the apparent danger of this practice. We have identified a nonspecific carboxyl-esterase that catalyzes the ethyl transesterification of cocaine to ethylcocaine in the presence of ethanol. In the absence of ethanol, this human liver esterase catalyzes the hydrolysis of cocaine to benzoylecgonine, a metabolite that is inactive as a psychomotor stimulant. A second human liver esterase is also described. This enzyme catalyzes hydrolysis of cocaine to ecgonine methyl ester, also inactive as a stimulant. These two liver esterases may play important roles in regulating the metabolic inactivation of cocaine.

Carboxylic Ester Hydrolases↗
PubMed1990

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.

Alcohol Dehydrogenase↗
PubMed1990

Alcohol and aldehyde dehydrogenase.

The enzymes mainly responsible for ethanol degradation in humans are liver alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH). Polymorphisms occur in both enzymes, with marked differences in the steady-state kinetic constants. The Km-values for ethanol of ADH isoenzymes relevant for alcohol degradation range from 49 microM to 36 microM, and the Vmax-values from 0.6 to 10 U/mg. Expression of an inactive form of the ALDH2 isoenzyme, the so-called Oriental variant, results in impaired acetaldehyde metabolizing capacity. The differences in ethanol and acetaldehyde metabolizing activities of allelic enzyme forms may be responsible in part for the large variation in the alcohol metabolism rate in humans. Interindividual differences in the isoenzyme pattern may contribute to the genetically determined predisposition for excessive alcohol intake.

Alcohol Dehydrogenase↗
PubMed1989

Purification and steady-state kinetic characterization of human liver beta 3 beta 3 alcohol dehydrogenase.

Human liver alcohol dehydrogenase catalyzes the NAD+-dependent oxidation of alcohols. Isoenzymes are produced in liver by five different genes, two of which are polymorphic. We have studied the three beta beta isoenzymes produced at ADH2 because they exhibit very different kinetic properties and they appear with different frequencies in different racial populations. The beta 3 beta 3 isoenzyme which appears in 25% of black Americans was purified to homogeneity, and conditions were found to stabilize this labile isoenzyme. The comparison of substrate specificity among beta beta isoenzymes for primary straight-chain alcohols indicates that there is a positive correlation between Vmax/KM and the log octanol/water partition coefficient for alcohols with beta 2 beta 2 and beta 3 beta 3 but not with beta 1 beta 1. Methyl substitutions at C1 or C2 of these alcohols reduce the catalytic efficiency with all three isoenzymes. The KM and Ki values of beta 3 beta 3 for NAD+ and NADH are substantially higher than values for beta 1 beta 1 or beta 2 beta 2. The Vmax of beta 3 beta 3 ethanol oxidation is 90 times that of beta 1 beta 1. Sequencing of the beta 3 subunit and gene indicates that the polymorphism results from a single amino acid exchange of Cys-369 in beta 3 for Arg-369 in beta 1 and beta 2 [Burnell et al. (1987) Biochem. Biophys. Res. Commun. 146, 1227-1233]. In horse alcohol dehydrogenase and beta 1 beta 1, the guanidino group of Arg-369 is thought to stabilize the NAD(H)-enzyme complex by bonding to one of the pyrophosphate oxygens.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1989

Stereospecific oxidation of secondary alcohols by human alcohol dehydrogenases.

The human liver alpha alpha alcohol dehydrogenase exhibits a different substrate specificity and stereospecificity for secondary alcohols than the human beta 1 beta 1, and gamma 1 gamma 1 or horse liver alcohol dehydrogenases. All of the enzymes efficiently oxidize primary alcohols, but alpha alpha oxidizes secondary alcohols far more efficiently than human beta 1 beta 1 and gamma 1 gamma 1 or horse liver alcohol dehydrogenase. Specifically, alpha alpha oxidizes four- and five-carbon secondary alcohols with efficiencies 0.06-2.2 times that of primary homologs and oxidizes these secondary alcohols with efficiencies up to 3 orders of magnitude greater than those of the three other isoenzymes. Whereas the human beta 1 beta 1, gamma 1 gamma 1 and horse isoenzymes show a distinct preference toward (S)-(+)-3-methyl-2-butanol, the alpha alpha isoenzyme prefers (R)-(-)-3-methyl-2-butanol. Computer-simulated graphics demonstrate that the horse subunit accommodates (S)-(+)-3-methyl-2-butanol within the active site much better than the opposite stereoisomer, primarily due to steric hindrance caused by Phe-93. Human alpha may accommodate (R)-(-)-3-methyl-2-butanol better than (S)-(+)-3-methyl-2-butanol because of close contacts between the latter and Thr-48. These observations suggest that substitutions at positions 93 and 48 in the active site of human liver alcohol dehydrogenase isoenzymes may determine their substrate specificity for secondary alcohols.

Alcohol Dehydrogenase↗
PubMed1989

Genotypes for aldehyde dehydrogenase deficiency and alcohol sensitivity. The inactive ALDH2(2) allele is dominant.

Many Orientals lack the mitochondrial aldehyde dehydrogenase (ALDH2) activity responsible for the oxidation of acetaldehyde produced during ethanol metabolism. These individuals suffer the alcohol-flush reaction when they drink alcoholic beverages. The alcohol-flush reaction is the result of excessive acetaldehyde accumulation, and the unpleasant symptoms tend to reduce alcohol consumption. The subunit of this homotetrameric enzyme was sequenced and the abnormality in the inactive enzyme shown to be a substitution of lysine for glutamate at position 487. We have used the polymerase chain reaction to determine the genotypes of 24 livers from Japanese individuals. Correlating genotype with phenotype leads to the conclusion that the allele (ALDH2(2)) encoding the abnormal subunit is dominant.

Aldehyde Dehydrogenase↗
PubMed1988

Genetic polymorphism of enzymes of alcohol metabolism and susceptibility to alcoholic liver disease.

Differences in the pharmacokinetics of alcohol absorption and elimination are, in part, genetically determined. There are polymorphic variants of the two main enzymes responsible for ethanol oxidation in liver, alcohol dehydrogenase and aldehyde dehydrogenase. The frequency of occurrence of these variants, which have been shown to display strikingly different catalytic properties, differs among different racial populations. Since the activity of alcohol dehydrogenase in liver is a rate-limiting factor for ethanol metabolism in experimental animals, it is likely that the type and content of the polymorphic isoenzyme subunit encoded at ADH2, beta-subunit, and at ADH3, the gamma-subunit, are contributing factors to the genetic variability in ethanol elimination rate. The recent development of methods for genotyping individuals at these loci using white cell DNA will allow us to test this hypothesis as well as any relationship between ADH genotype and the susceptibility to alcoholism or alcohol-related pathology. A polymorphic variant of human liver mitochondrial aldehyde dehydrogenase, ADLH2, which has little or no acetaldehyde oxidizing activity has been identified. Individuals with the deficient ALDH2 phenotype do not have altered ethanol elimination rates but they do exhibit high blood acetaldehyde levels and dysphoric symptoms such as facial flushing, nausea and tachycardia, after drinking alcohol. Because acetaldehyde is so reactive, it binds to free amino groups of proteins including a 37 kilodalton hepatic protein-acetaldehyde adduct and may elicit an antibody response. We would predict that individuals who have low ALDH2 activity because of liver disease or because they have the inactive ALDH2 variant isoenzyme might form more protein-acetaldehyde adducts and elicit a greater immune response. These adducts may represent good biological markers of alcohol abuse and may also play a role in liver injury due to chronic alcohol consumption.

Alcohol Dehydrogenase↗
PubMed1988

Genotyping of human alcohol dehydrogenases at the ADH2 and ADH3 loci following DNA sequence amplification.

Humans are polymorphic at two of the alcohol dehydrogenase (ADH) loci important in ethanol metabolism, ADH2 and ADH3. Although the coding regions of these genes are 94% identical, they produce subunits that differ greatly in kinetic properties in vitro. These differences are likely to be reflected in the pharmacokinetics of alcohol metabolism, but studies have been hampered by the need to use liver biopsy specimens to determine the ADH phenotype. This problem has now been overcome by determining the genotype at these loci using DNA that has been amplified in vitro by the polymerase chain reaction. We report here the identification of all three of the ADH2 alleles and both of the ADH3 alleles. Any pair of ADH2 or ADH3 alleles can be distinguished using allele-specific oligonucleotide probes directed at their single base pair difference. In addition, ADH2(2) can be distinguished from ADH2(1) and ADH2(3) by detecting a new MaeIII site created in the third exon by the single base pair alteration in ADH2(2).

Alcohol Dehydrogenase↗
PubMed1987

The human beta 3 alcohol dehydrogenase subunit differs from beta 1 by a Cys for Arg-369 substitution which decreases NAD(H) binding.

The beta 3 beta 3 (formerly called beta Indianapolis) and beta 1 beta 1 isoenzymes of human alcohol dehydrogenase differ substantially in their catalytic properties. Specifically, the KM value for NAD+ of beta 3 beta 3 is 70 times greater than that of beta 1 beta 1, and the Ki value for NADH is 35 times greater than that of beta 1 beta 1. To identify the structural basis of these catalytic differences, we sequenced regions of the beta 3 subunit and the beta 3 gene. beta 3 differs from beta 1 by the substitution of Cys for Arg-369. Based on x-ray crystallography of horse ADH, Arg-369 should interact with the nicotinamide phosphate moiety of NAD(H). The Cys for Arg-369 substitution would decrease the enzyme's affinity for coenzyme and, thus, account for the observed kinetic differences between beta 3 beta 3 and beta 1 beta 1.

Alcohol Dehydrogenase↗
PubMed1987

Purification and characterization of the Danish (Skive) variant of mouse liver alcohol dehydrogenase.

The partially inbred Danish (Skive) strain of mice exhibits a form of liver alcohol dehydrogenase (ADH) which differs in electrophoretic mobility from that of all other inbred mouse strains thus far examined, e.g., C57BL/10, DBA/2J, and BALB/c. In order to compare the catalytic and molecular properties of the "variant" and "normal" enzyme forms, they were purified to homogeneity by ion-exchange and affinity chromatography. Tryptic peptides of reduced and carboxymethylated subunits of the normal and variant ADH forms were mapped by thin-layer two-dimensional electrophoresis and chromatography and by reversed-phase high-performance liquid chromatography. A unique nonapeptide in the Danish mouse liver ADH which did not appear in enzymes from C57BL/10, DBA/2J, or BALB/c mice was identified by both methods. Amino acid sequencing of this peptide revealed that the Arg residue at position 124, as predicted from the cDNA sequence of ADH in DBA/2J mice, has been replaced by Leu in the Danish variant. The Leu for Arg substitution in the variant form appears to account for its decreased cathodic mobility with electrophoresis in starch gels at pH 7.2. The Km and Vmax of ADH from the Danish strain for three primary alcohols and three aldehydes were similar in value to those of ADH from the C57BL/10, DBA/2J, and BALB/c strains. Based on the X-ray structure of horse liver ADH, position 124 is on the solvent-exposed surface of the catalytic domain. The finding that the kinetic constants are similar for the normal and variant forms is consistent with the observation that this residue is not in the active site and that there is no known role for it in the ADH catalytic mechanism.

Alcohol Dehydrogenase↗
PubMed1987

Ethanol metabolism.

Alcohol is metabolized by two pathways in humans: the ADH pathway which accounts for the bulk of the metabolism, and the MEOS pathway which contributes to the increased rate of ethanol elimination at high blood alcohol levels. The increased rate of elimination which results from chronic alcohol consumption is due to an increase in MEOS activity. The activities of these pathways are influenced by environmental factors such as smoking, diet, and endocrine factors. In addition, individuals inherit different types of ADH isoenzymes which have different kinetic properties. Individuals with different phenotypic variants, e.g. the beta 1 vs beta 2 isoenzymes, appear to have different rates of ethanol elimination. The cloning of the ADH genes and the availability of molecular hybridization methods now make it possible to genotype individuals and to correlate the genotype with both alcohol elimination rates and with the risk of developing medical complications of alcoholism or even of developing alcoholism itself.

Alcohol Dehydrogenase↗
PubMed1987

Structure of the mouse Adh-1 gene and identification of a deletion in a long alternating purine-pyrimidine sequence in the first intron of strains expressing low alcohol dehydrogenase activity.

The ethanol-active alcohol dehydrogenase (ADH-A2) expressed at high levels in mouse liver is encoded by the Adh-1 gene. Inbred strains differ in the amount of ADH-A2 expressed. We report here the cloning and sequencing of the Adh-1 genes from mouse strains that express high and low amounts of ADH-A2 in liver (strains YBR/Ki and Balb/c respectively). The gene contains nine exons, and encodes an ADH-A subunit identical to that encoded by the cDNA isolated from DBA/2J, a strain with low liver ADH activity. This demonstrates that the difference between strains in liver ADH activity is not due to differences in the amino acid sequence of the ADH-A2. The 5'-nontranslated region and at least the first 225 bp 5' to the transcriptional start point are identical in both strains. We have found restriction fragment length polymorphisms in the Adh-1 gene that correlate with the level of expression of ADH-A2 in different strains. One of these RFLPs is within a remarkably long (288 bp) strictly alternating purine-pyrimidine sequence located in the first intron. This region in YBR/Ki contains 25 copies of the sequence ATGT(A/G)T (four of them inverted), which closely resembles important elements in the SV40 enhancer region. Balb/c mice, which express Adh-1 at lower levels, have a deletion that removes 101 bp of this sequence and also have several transition mutations; the comparable region has nine fewer ATGT(A/G)T repeats. These results suggest that the difference in gene expression may be due to differences in these hexamers or in other portions of the alternating purine-pyrimidine sequences, rather than in cis-acting sequences in the proximal 5' (promoter) region.

Alcohol Dehydrogenase↗
PubMed1987

Purification of human liver aldehyde dehydrogenase by high-performance liquid chromatography and identification of isoenzymes by immunoblotting.

Human liver aldehyde dehydrogenase (ALDH) exists in multiple molecular forms. Two different isoenzymes of ALDH have been purified which will oxidize acetaldehyde to acetate. ALDH1 is localized principally in hepatocyte cytosol and exhibits a Km for acetaldehyde of about 0.1 mM at pH 9.5. ALDH2 is mitochondrial in origin and exhibits low Km for acetaldehyde, about 1 microM. We have developed rapid purification procedures for ALDH1 and ALDH2 by use of agarose-AMP affinity chromatography and high-performance anion-exchange liquid chromatography (HPLC). The method takes less time and affords higher yields of the labile ALDH isoenzymes than conventional column chromatography methods. A previously uncharacterized ALDH form has been identified by anion-exchange HPLC which exhibits high Km for acetaldehyde, about 1 mM, and is very labile. Polyclonal antibodies to the purified ALDH1 and ALDH2 isoenzymes have been prepared. As evidenced by immunoblotting of starch gels containing the purified isoenzymes, anti-ALDH1 does not crossreact with ALDH2 and anti-ALDH2 does not crossreact with ALDH1. The anti-ALDH2 antibody identifies the "inactive" variant of ALDH2 in Japanese livers exhibiting the "deficient" ALDH2 phenotype. The sensitivity of detection of ALDH isoenzymes in liver homogenate-supernatants by immunoblotting of starch gels is about 10-fold greater than that by activity staining.

Aldehyde Dehydrogenase↗