Kinetic effects of a single-amino acid mutation in a highly variable loop (residues 114-120) of class IV ADH.
Class IV alcohol dehydrogenase shows a deletion at position 117 with respect to class I enzymes, which typically have a Gly residue. In class I structures, Gly117 is part of a loop (residues 114-120) that is highly variable within the alcohol dehydrogenase family. A mutant human class IV enzyme was engineered in which a Gly residue was inserted at position 117 (G117ins). Its kinetic properties, regarding ethanol and primary aliphatic alcohols, secondary alcohols and pH profiles, were determined and compared with the results obtained in previous studies in which the size of the 114-120 loop was modified. For the enzymes considered, a smaller loop was associated with a lower catalytic efficiency towards short-chain alcohols (ethanol and propanol) and secondary alcohols, as well as with a higher K(m) for ethanol at pH 7.5 than at pH 10.0. The effect can be rationalized in terms of a more open, solvent-accessible active site in class IV alcohol dehydrogenase, which disfavors productive binding of ethanol and short-chain alcohols, specially at physiological pH.
Allali-Hassani
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Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain
Crosas
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Parés
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Farrés
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D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000438:Alcohols / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D000431:Ethanol / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000438:Alcohols; D011994:Recombinant Proteins; D009243:NAD; D000431:Ethanol; D000426:Alcohol Dehydrogenase; C105552:alcohol dehydrogenase IV