Role of arginine 115 in fatty acid activation and formaldehyde dehydrogenase activity of human class III alcohol dehydrogenase.
Modification of class III alcohol dehydrogenase (chi chi-ADH) with phenylglyoxal eliminates fatty acid activation by pentanoate and octanoate and concomitantly increases specific activity toward ethanol and 3-methylcrotyl alcohol 2-3-fold. In contrast, chemical modification decreases activity toward S-(hydroxymethyl)glutathione (FDH activity) and 12-hydroxydodecanoic acid by increasing Km, pointing to a role for arginine in binding anionic substrates. Modification with [7-14C]phenylglyoxal indicates that only one arginine residue per subunit is modified. Sequence analysis of tryptic peptides indicates that Arg-115 is modified. Site-directed mutation of this residue to alanine eliminates both fatty acid activation and FDH activity, thus confirming the identity of the modified residue and its function. These results account in part for the unique specificity of chi chi-ADH relative to other human ADH isozymes.
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
B
B
Holmquist
J M
JM
Moulis
K
K
Engeland
B L
BL
Vallee
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010658:Phenylglyoxal / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D005227:Fatty Acids; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D001120:Arginine; D000426:Alcohol Dehydrogenase; C020145:formaldehyde dehydrogenase (glutathione); D000445:Aldehyde Oxidoreductases; D014357:Trypsin; D010658:Phenylglyoxal