Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation.
The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. The activities of the recombinant native and mutant enzymes toward ethanol are essentially identical, but mutagenesis greatly decreases the kcat/Km values for glutathione-dependent formaldehyde oxidation. The catalytic efficiency for the Asp variant is < 0.1% that of the unmutated enzyme, due to both a higher Km and a lower kcat value. As with the native enzyme, neither mutant can oxidize methanol, be saturated by ethanol, or be inhibited by 4-methylpyrazole; i.e., they retain these class III characteristics. In contrast, however, their activation by fatty acids, another characteristic unique to class III alcohol dehydrogenase, is markedly attenuated. The Ala mutant is activated only slightly, but the Asp mutant is not activated at all. The results strongly indicate that Arg-115 in class III alcohol dehydrogenase is a component of the binding site for activating fatty acids and is critical for the binding of S-hydroxymethylglutathione in glutathione-dependent formaldehyde dehydrogenase activity.
Engeland
K
K
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, MA 02115.
Höög
J O
JO
Holmquist
B
B
Estonius
M
M
Jörnvall
H
H
Vallee
B L
BL
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005227:Fatty Acids; D007527:Isoenzymes; D011994:Recombinant Proteins; D001120:Arginine; D000426:Alcohol Dehydrogenase; C020145:formaldehyde dehydrogenase (glutathione); D000445:Aldehyde Oxidoreductases