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

• Published in: Biochemistry (Easton) Vol. 30; no. 4; pp. 1062 - 1068
• Main Authors: Ehrig, Torsten, Hurley, Thomas D, Edenberg, Howard J, Bosron, William F
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.01.1991
• Subjects: alcohol dehydrogenase; Alcohol Dehydrogenase - chemistry; Alcohol Dehydrogenase - genetics; Analytical, structural and metabolic biochemistry; Binding, Competitive; Biological and medical sciences; Catalysis; Decanoic Acids - pharmacology; Enzymes and enzyme inhibitors; Ethanol - metabolism; Fundamental and applied biological sciences. Psychology; Glutamine - genetics; histidine; Histidine - genetics; horses; Humans; Isoenzymes - genetics; Kinetics; liver; Liver - drug effects; Liver - enzymology; mechanisms; Mutagenesis; NAD - metabolism; Oxidoreductases; substitution; Trifluoroethanol - pharmacology; Human; Histidine; Isoelectrical focusing; Mutagenesis; Enzyme; Liver; Active site; Alcohol dehydrogenase; Kinetics; Crystallography; X ray diffraction; Spatial structure
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00218a026

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.