Activation and inactivation of horse liver alcohol dehydrogenase with pyridoxal compounds.
Pyridoxal compounds can either activate or inactivate horse liver alcohol dehydrogenase in differential labeling experiments. Amino groups outside of the active sites were modified with ethyl acetimidate, while the amino groups in the active sites were protected by the formation of the complex with NAD-plus and pyrazole. After removal of the NAD-plus and pyranzole, the partially acetimidylated enzyme was reductively alkylated with pyridoxal and NaBH4, with the incorporation of one pyridoxal group per subunit of the enzyme. The turnover numbers for the reaction of NAD-plus and ethanol increased by 15-fold, and for NADH and acetaldehyde by 32-fold. The Michaelis and inhibition constants increased 80-fold or more. Pyridoxal phosphate and NaBH4 also modified one group per subunit, but the turnover numbers decreased by 10-fold and the kinetic constants were intermediate between those obtained for pyridoxyl alcohol dehydrogenase and the partially acetimidylated enzyme. With native enzyme, the rates of dissociation of the enzyme-coenzyme complexes are rate-limiting in the catalytic reactions. The pyridoxyl enzyme is activated because the rates of dissociation of the enzyme-coenzyme complexes are increased. The rates of binding of coenzyme to phosphopyridoxyl enzyme have decreased due to the introduction of the negatively charged phosphate. The size of the group is not responsible for this decrease since these rates are not greatly decreased by the incorporation of pyridoxal. For both pyrodoxal and phosphopyridoxyl alcohol dehydrogenases, the interconversion of the ternary complex is at least partially rate-limiting. Chymotryptic-tryptic digestion of pryidoxyl enzyme produced a major peptide corresponding to residues 219 to 229, in which Lys 228 had reacted with pyridoxal. The same lysine residue reacted with pyridoxal phosphate.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D002918:Chymotrypsin; D003903:Deuterium; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011730:Pyridoxal / Q000494:pharmacology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin
D000596:Amino Acids; D010446:Peptide Fragments; D000431:Ethanol; D011730:Pyridoxal; D011732:Pyridoxal Phosphate; D003903:Deuterium; D000429:Alcohol Oxidoreductases; D002918:Chymotrypsin; D014357:Trypsin