Oxidative inactivation of an extramitochondrial acetyl-CoA hydrolase by autoxidation of L-ascorbic acid.
The activity of acetyl-CoA hydrolase (dimeric form) purified from the supernatant fraction of rat liver was shown to have a half-life (t1/2) of 3 min at 0 degree C, but to stable at 37 degrees C (t1/2 = 34 h) [Isohashi, F., Nakanishi, Y. & Sakamoto, Y. (1983) Biochemistry 22, 584-590]. Incubation of the purified enzyme with L-ascorbic acid (AsA) at 37 degrees C resulted in inactivation of the enzyme (t1/2 = 90 min at 2 mM AsA). The extent of inactivation was greatly enhanced by addition of transition metal ions (Cu2+, Fe2+, and Fe3+). Thiol reducing agents, such as reduced glutathione and DL-dithiothreitol, protected the hydrolase from inactivation by AsA. However, these materials did not restore the catalytic activity of the enzyme inactivated by AsA. When AsA solution containing Cu2+ was preincubated under aerobic conditions at 37 degrees C for various times in the absence of enzyme, and then aliquots were incubated with the enzyme solution for 20 min, remaining activity was found to decrease with increase in the preincubation time, reaching a minimum at 60 min. However, further preincubation reduced the potential for inactivation. Catalase, a hydrogen peroxide (H2O2) scavenger, almost completely prevented inactivation of the enzyme by AsA plus Cu2+. Superoxide dismutase and tiron, which are both superoxide (O2-) scavengers, also prevented inactivation of the enzyme. A high concentration of mannitol, a hydroxyl radical (OH) scavenger, partially protected the enzyme from inactivation. These results suggest that inactivation of the enzyme by AsA in the presence of Cu2+ was due to the effect of active oxygen species (H2O2, O2-, OH) that are known to be autoxidation products of AsA. Valeryl-CoA, a competitive inhibitor of acetyl-CoA hydrolase, greatly protected the enzyme from inactivation by AsA plus Cu2+, but ATP and ADP, which are both effectors of this enzyme, had only slight protective effects. These results suggest that inactivation of this enzyme by addition of AsA plus Cu2+ was mainly due to attack on its active site.
Nakanishi
Y
Y
Isohashi
F
F
Matsunaga
T
T
Sakamoto
Y
Y
D000104:Acetyl-CoA Hydrolase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism / Q000494:pharmacology*; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D005609:Free Radicals; D008099:Liver / Q000201:enzymology; D008297:Male; D008670:Metals / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D051381:Rats; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors*; D014969:Xanthine Oxidase / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism
D002614:Chelating Agents; D005609:Free Radicals; D008670:Metals; D014970:Xanthines; D003300:Copper; D014969:Xanthine Oxidase; D013869:Thiolester Hydrolases; D000104:Acetyl-CoA Hydrolase; D001205:Ascorbic Acid