Inversion of the substrate specificity of yeast alcohol dehydrogenase.
The relationship between the size of the substrate binding pocket and the catalytic reactivities with varied alcohols was studied with the Saccharomyces cerevisiae alcohol dehydrogenase I (ScADH) and compared with the liver enzymes from horse (EqADH, EE isoenzyme) and monkey (MmADH alpha, alpha-isoenzyme). The yeast enzyme is most active with ethanol, and its activity decreases as the size of the alcohol is increased, whereas the activities of the liver enzymes increase with larger alcohols. The substrate pocket in ScADH was enlarged by single substitutions of Thr-48 to Ser (T48S), Trp-57 to Met (W57M), and Trp-93 to Ala (W93A), and a double change, T48S:W93A, and a triple, T48S:W57M:W93A. The T48S enzyme has the same pattern of activity (V/K) as wild-type ScADH for linear primary alcohols. The W57M enzymes have lowered reactivity with primary and secondary alcohols. The W93A and T48S:W93A enzymes resemble MmADH alpha in having an inverted specificity pattern for primary alcohols, being 3- and 10-fold more active on hexanol and 350- and 540-fold less active on ethanol, and are as reactive as the liver enzymes with long chain primary alcohols. The three Ala-93 enzymes also acquired weak activity on branched chain alcohols and cyclohexanol.
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Department of Biochemistry, University of Iowa, Iowa City 52242.
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D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003511:Cyclohexanols / Q000378:metabolism; D003903:Deuterium; D000882:Haplorhini; D006736:Horses; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D003511:Cyclohexanols; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D003903:Deuterium; D000444:Aldehyde Dehydrogenase