Residues specific for class III alcohol dehydrogenase. Site-directed mutagenesis of the human enzyme.
Human class III alcohol dehydrogenase (with both glutathione-dependent formaldehyde dehydrogenase and alcohol dehydrogenase activities) was expressed, and studied by site-directed mutagenesis corresponding to three amino acid residues that are affecting the substrate-binding pocket of class I (with alcohol dehydrogenase activity only). A Thr48Ala exchange results in an enzyme essentially without alcohol dehydrogenase activity but with some glutathione-dependent formaldehyde dehydrogenase activity retained. This indicates that coordination to the enzyme of S-hydroxymethylglutathione is mediated by interactions additional to, or different from, those utilized for primary and secondary alcohols. An Asp57Leu mutation causes considerable loss of the formaldehyde dehydrogenase activity, showing that a negative charge at position 57 is a prerequisite for this class III-type of activity, in the same manner as a positive charge at position 115 has been previously demonstrated to be crucial. Therefore, Asp57 and Arg115 appear to contribute equally to the interactions with S-hydroxymethylglutathione, compatible with defining the class III-type of specificity and possibly explaining the dependence on glutathione. A Tyr93Phe mutant exhibits decreased kcat values for substrates in general and correlates with inhibition of alcohol dehydrogenase activity by 4-methylpyrazole, a potent inhibitor of the class I enzymes. In a double mutant, Asp57Leu/Tyr93Phe, the effects of the two mutations are potentiating one another, yielding a fall in kcat/Km for hydroxymethylglutathione by a factor of 1250, i.e., a still further loss of class III-type activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Estonius
M
M
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Höög
J O
JO
Danielsson
O
O
Jörnvall
H
H
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001216:Asparagine; D001483:Base Sequence; D001665:Binding Sites; D000077604:Fomepizole; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010649:Phenylalanine; D011720:Pyrazoles / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine
D011720:Pyrazoles; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D001216:Asparagine; D000077604:Fomepizole; D000426:Alcohol Dehydrogenase; D007930:Leucine