Oxidative inactivation of an extramitochondrial acetyl-CoA hydrolase by autoxidation of l-ascorbic acid
Open Access
- 1 October 1985
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 152 (2) , 337-342
- https://doi.org/10.1111/j.1432-1033.1985.tb09203.x
Abstract
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°C, but to be stable at 37°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°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°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 result suggest that inactivation of the enzyme by AsA in the presence of Cu2+ was due to the effect of active oxygen species (H2O2, O−2, 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.This publication has 27 references indexed in Scilit:
- A cold‐labile acetyl‐coenzyme‐A hydrolase from the supernatant fraction of rat liverEuropean Journal of Biochemistry, 1984
- Factors Affecting the Cold Inactivation of an Acetyl‐Coenzyme‐A Hydrolase Purified from the Supernatant Fraction of Rat LiverEuropean Journal of Biochemistry, 1983
- Effects of nucleotides on a cold labile acetyl-coenzyme A hydrolase from the supernatant fraction of rat liverBiochemistry, 1983
- An improved method for determination of l-ascorbic acid and l-dehydroascorbic acid in blood plasmaClinica Chimica Acta; International Journal of Clinical Chemistry, 1980
- The Biology of Oxygen RadicalsScience, 1978
- Acetyl‐CoA Hydrolase; Activity, Regulation and Physiological, Significance of the Enzyme in Brown Adipose Tissue from HamsterEuropean Journal of Biochemistry, 1976
- Utilization of volatile fatty acids in ruminants. IV. Relative activities of acetyl CoA synthetase and acetyl CoA hydrolase in mitochondria and intracellular localization of acetyl CoA synthetaseJournal of Agricultural and Food Chemistry, 1972
- The Preparation of S-Succinyl Coenzyme AJournal of the American Chemical Society, 1953
- The Reaction between Ferrous Iron and Peroxides. II. Reaction with Hydrogen Peroxide, in the Presence of OxygenJournal of the American Chemical Society, 1949
- Oxidation Processes. XVI.1 The Autoxidation of Ascorbic AcidJournal of the American Chemical Society, 1943