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

Burnell, Joe C.; Li, Ting Kai; Bosron, William F.

doi:10.1021/bi00443a005

Cited by 40 publications

(23 citation statements)

References 33 publications

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“…These yields are similar to those obtained for ␤ 1 (14). The specific activity of purified recombinant ␤ 3 at 2.5 mM NAD ϩ and 66 mM ethanol, 3.8 units/mg, was higher than that of purified ␤ 3 from human liver obtained at autopsy, 2.8 units/mg (11) (Table I). …”

Section: Resultssupporting

confidence: 81%

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X-ray Structure of Human β3β3 Alcohol Dehydrogenase

Davis

Bosron

Stone

et al. 1996

Journal of Biological Chemistry

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The three-dimensional structure of the human ␤ 3 ␤ 3 dimeric alcohol dehydrogenase (␤ 3 ) was determined to 2.4-Å resolution. ␤ 3 was crystallized as a ternary complex with the coenzyme NAD ؉ and the competitive inhibitor 4-iodopyrazole. ␤ 3 is a polymorphic variant at ADH2 that differs from ␤ 1 by a single amino acid substitution of Arg-369 3 Cys. The available x-ray structures of mammalian alcohol dehydrogenases show that the side chain of Arg-369 forms an ion pair with the NAD(H) pyrophosphate to stabilize the E⅐NAD(H) complex. The Cys-369 side chain of ␤ 3 cannot form this interaction. The three-dimensional structures of ␤ 3 and ␤ 1 are virtually identical, with the exception that Cys-369 and two water molecules in ␤ 3 occupy the position of Arg-369 in ␤ 1 . The two waters occupy the same positions as two guanidino nitrogens of Arg-369. Hence, the number of hydrogen bonding interactions between the enzyme and NAD(H) are the same for both isoenzymes. However, ␤ 3 differs from ␤ 1 by the loss of the electrostatic interaction between the NAD(H) pyrophosphate and the Arg-369 side chain. The equilibrium dissociation constants of ␤ 3 for NAD ؉ and NADH are 350-fold and 4000-fold higher, respectively, than those for ␤ 1 . These changes correspond to binding free energy differences of 3.5 kcal/mol for NAD ؉ and 4.9 kcal/mol for NADH. Thus, the Arg-369 3 Cys substitution of ␤ 3 isoenzyme destabilizes the interaction between coenzyme and ␤ 3 alcohol dehydrogenase.Alcohol dehydrogenase (ADH, EC 1.1.1.1) 1 catalyzes the rate-limiting step in the oxidation of ethanol (1, 2). Human ADH isoenzymes are dimeric enzymes that contain one structural and one catalytic zinc per subunit. Human ADH isoenzyme subunits have between 373 and 379 amino acids and are encoded by the ADH1 (␣), ADH2 (␤), ADH3 (␥), ADH4 (), ADH5 (␥), ADH6, and ADH7 () genes (3, 4). The ␣, ␤, and ␥ ADH subunits share greater than 93% sequence identity and form a complex group of homodimeric and heterodimeric isoenzymes.2 These isoenzymes have relatively high catalytic efficiency for ethanol oxidation and account for the majority of ethanol oxidation by the liver (3,5,6).Polymorphism at the ADH2 gene gives rise to the ␤ 1 , ␤ 2 , and ␤ 3 subunits (3, 4). These polymorphic variants are the result of single amino acid substitutions. Arg-47 is substituted by His in ␤ 2 , and Arg-369 is substituted by Cys in ␤ 3 (7, 8). The ␤ 2 (ADH2*2) and ␤ 3 (ADH2*3) alleles are observed in about 65% of Japanese and Chinese, and 25% of African-Americans, respectively (5, 9). The three human ␤ isoforms have considerably different steady-state kinetic properties (10). The ␤ 3 isoenzyme exhibits a 50-fold greater K m for NAD ϩ , an 100-fold greater V max for ethanol oxidation, and a decreased pH optimum for ethanol oxidation, relative to ␤ 1 (Table I) (11).Three-dimensional structures have been determined for horse liver EE ADH as an apoenzyme and complexed with a variety of substrates or inhibitors (12, 13). Structures of ␤ 1 complexed with NAD(H) and cyclohexanol, ␤ 2 complexed ...

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Section: Resultssupporting

confidence: 81%

“…The three human ␤ isoforms have considerably different steady-state kinetic properties (10). The ␤ 3 isoenzyme exhibits a 50-fold greater K m for NAD ϩ , an 100-fold greater V max for ethanol oxidation, and a decreased pH optimum for ethanol oxidation, relative to ␤ 1 (Table I) (11).…”

mentioning

confidence: 99%

X-ray Structure of Human β3β3 Alcohol Dehydrogenase

Davis

Bosron

Stone

et al. 1996

Journal of Biological Chemistry

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“…ADHs are very abundant in liver and play a major role in hepatic ethanol metabolism [22,23,25,46]. They are also capable of metabolizing longer-chain and cyclic alcohols, however, primary alcohols seem to be their preferred substrates [22,23].…”

Section: Resultsmentioning

confidence: 99%

Release and uptake of volatile organic compounds by human hepatocellular carcinoma cells (HepG2) in vitro

et al. 2013

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BackgroundVolatile organic compounds (VOCs) emitted by human body offer a unique insight into biochemical processes ongoing in healthy and diseased human organisms. Unfortunately, in many cases their origin and metabolic fate have not been yet elucidated in sufficient depth, thus limiting their clinical application. The primary goal of this work was to identify and quantify volatile organic compounds being released or metabolized by HepG2 hepatocellular carcinoma cells.MethodsThe hepatocellular carcinoma cells were incubated in specially designed head-space 1-L glass bottles sealed for 24 hours prior to measurements. Identification and quantification of volatiles released and consumed by cells under study were performed by gas chromatography with mass spectrometric detection (GC-MS) coupled with head-space needle trap device extraction (HS-NTD) as the pre-concentration technique. Most of the compounds were identified both by spectral library match as well as retention time comparison based on standards.ResultsA total of nine compounds were found to be metabolised and further twelve released by the cells under study (Wilcoxon signed-rank test, p<0.05). The former group comprised 6 aldehydes (2-methyl 2-propenal, 2-methyl propanal, 2-ethylacrolein, 3-methyl butanal, n-hexanal and benzaldehyde), n-propyl propionate, n-butyl acetate, and isoprene. Amongst the released species there were five ketones (2-pentanone, 3-heptanone, 2-heptanone, 3-octanone, 2-nonanone), five volatile sulphur compounds (dimethyl sulfide, ethyl methyl sulfide, 3-methyl thiophene, 2-methyl-1-(methylthio)- propane and 2-methyl-5-(methylthio) furan), n-propyl acetate, and 2-heptene.ConclusionsThe emission and uptake of the aforementioned VOCs may reflect the activity of abundant liver enzymes and support the potential of VOC analysis for the assessment of enzymes function.

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“…The ADH2 and ADH3 loci are both polymorphic. ADH2 encodes for the b1 (``usual''), b2 (``atypical'') and b3 subunit 15,16,17,18,19 . The ADH3 locus encodes for the g1 and g2 subunits.…”

Section: Alcohol Dehydrogenasementioning

confidence: 99%

Genetic Polymorphisms in Ethanol Metabolism: Issues and Goals for Physiologically Based Pharmacokinetic Modeling*

Pastino¹,

Flynn²,

Sultatos³

2000

Drug and Chemical Toxicology

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Chronic exposure to excessive ethanol consumption has adverse effects on virtually all organs and tissues in the body, including but not limited to the liver, pancreas, reproductive organs, central nervous system, and the fetus. Exposure to ethanol can also enhance the toxicity of other chemicals. Not all persons exposed to the same amount of ethanol experience the same degree of adverse effects. For example, only 12% to 13% of alcohol abusers develop cirrhosis. Possible factors which may alter susceptibility include age, sex, nutritional status, health status (i.e., smokers) and race. Some of these factors affect susceptibility because they alter ethanol metabolism, which occurs primarily in the liver by alcohol dehydrogenase (ADH). Genetic polymorphisms for ADH partially account for the observed differences in ethanol elimination rates among various populations but the relative contribution to susceptibility is not completely understood. Incorporation of the kinetic parameters associated with ADH polymorphisms into a physiologically based pharmacokinetic (PBPK) model for ethanol will aid in assessing the relative contribution to susceptibility. The specific information required to develop this model includes Km and Kcat values for each ADH isoform and the amount of each isoform present in the liver. Blood ethanol concentrations (BEC) from various populations with known ADH phenotypes are also necessary to validate the model. The impact of inclusion of these data on PBPK model predictions was examined using available information from adult white and African American males.

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

Cited by 40 publications

References 33 publications

X-ray Structure of Human β3β3 Alcohol Dehydrogenase

X-ray Structure of Human β3β3 Alcohol Dehydrogenase

Release and uptake of volatile organic compounds by human hepatocellular carcinoma cells (HepG2) in vitro

Genetic Polymorphisms in Ethanol Metabolism: Issues and Goals for Physiologically Based Pharmacokinetic Modeling*

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