Identification of the lysine residue modified during the activation of acetimidylation of horse liver alcohol dehydrogenase.
A single amino group in horse liver alcohol dehydrogenase was modified with methyl(14C)acetimidate by a differential labeling procedure. Lysine residues outside the active site were modified with ethyl acetimidate while a lysine residue in the active site was protected by the formation of an enzyme-NAD+-pyrazole complex. After the protecting reagents were removed, the enzyme was treated with methyl(14C)acetimidate. Enzyme activity was enhanced 13-fold as 1.1 (14C)acetimidyl group was incorporated per active site. A labeled peptide was isolated from a tryptic-chymotryptic digest of the modified enzyme in 35% overall yield. Amino acid composition and sequential Edman degradations identified the peptide as residues 219-229; lysine residue 228 was modified with the radioactive acetimidyl group.
D000085:Acetates / Q000494:pharmacology*; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002918:Chymotrypsin; D006736:Horses; D007094:Imides / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin
D000085:Acetates; D000596:Amino Acids; D007094:Imides; D010446:Peptide Fragments; D000429:Alcohol Oxidoreductases; D002918:Chymotrypsin; D014357:Trypsin; D008239:Lysine