Sulfhydryl groups of an extramitochondrial acetyl-CoA hydrolase from rat liver.
An extramitochondrial acetyl-CoA hydrolase (EC 3.1.2.1) purified from rat liver was inactivated by heavy metal cations (Hg2+, Cu2+, Cd2+ and Zn2+), which are known to be highly reactive with sulfhydryl groups. Their order of potency for enzyme inactivation was Hg2+ greater than Cu2+ greater than Cd2+ greater than Zn2+. This enzyme was also inactivated by various sulfhydryl-blocking reagents such as p-hydroxymercuribenzoate (PHMB), N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and iodoacetate (IAA). DL-Dithiothreitol (DTT) reversed the inactivation of this enzyme by DTNB markedly, and that by PHMB slightly, but did not reverse the inactivations by NEM, DTNB and IAA. Benzoyl-CoA (a substrate-like competitive inhibitor) and ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by PHMB, NEM, DTNB and IAA. These results suggest that the essential sulfhydryl groups are on or near the substrate binding site and nucleotide binding site. The enzyme contained about four sulfhydryl groups per mol of monomer, as estimated with DTNB. When the enzyme was denatured by 4 M guanidine-HCl, about seven sulfhydryl groups per mol of monomer reacted with DTNB. Two of the four sulfhydryl groups of the subunit of the native enzyme reacted with DTNB first without any significant inactivation of the enzyme, but its subsequent reaction with the other two sulfhydryl groups seemed to be involved in the inactivation process.
Nakanishi
Y
Y
Department of Oncology, Osaka University Medical School, Japan.
Isohashi
F
F
Ebisuno
S
S
Sakamoto
Y
Y
D000104:Acetyl-CoA Hydrolase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D004229:Dithiothreitol / Q000494:pharmacology; D004793:Enzyme Reactivators; D008099:Liver / Q000201:enzymology*; D008297:Male; D008670:Metals / Q000494:pharmacology; D051381:Rats; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013869:Thiolester Hydrolases / Q000032:analysis*
D004793:Enzyme Reactivators; D008670:Metals; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D013869:Thiolester Hydrolases; D000104:Acetyl-CoA Hydrolase; D004229:Dithiothreitol