Oxidative inactivation of an extramitochondrial acetyl-CoA hydrolase by autoxidation of l-ascorbic acid

Abstract

The activity of acetyl-CoA hydrolase (dimeric form) purified from the supernatant fraction of rat liver was shown to have a half-life ( t 1 / 2 ) of 3 min at 0"C, but to be stable at 3 7 T (t1/2 = 34 h) Biochemistry 22, 584-5901, Incubation of the purified enzyme with L-ascorbic acid (AsA) at 3 7~' C resulted in inactivation of the enzyme ( t l j z = 90 min at 2 mM AsA). The extent of inactivation was greatly enhanced by addition of transition metal ions (Cu2+, Fez', and Fe3+). Thiol reducing agents, such as reduced glutathione and rx-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 Cuz+ 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 incrcase in the preincubation time, reaching a minimum at 60 min. However, further preincubation reduced the potential for inactivation. Catalase, a hydrogen peroxide ( H 2 0 2 ) scavenger, almost completely prevented inactivation of the enzyme by AsA plus Cu2+. Superoxide dismutase and tiron, which are both superoxide (0;) 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 ( H z 0 2 , O;, 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 CU", but ATP and ADP, which are both effectors ofthis 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.Acetyl-CoA hydrolase has been found in a wide variety of tissues of many species [l -51. In rats, extramitochondrial acetyl-CoA hydrolase is extremely labile in a liver homogenate [5] as well as in excised liver [6] prepared a t low temperature. The enzyme purified from the supernatant fraction of rat liver is activated by ATP and completely inhibited by ADP [5] and has various forms (tetramer, dimer and monomer) [7]. In the presence of ATP or ADP, but not AMP, at room temperature, the dimeric form ( M , 135000) of the enzyme reassociates to the tetraineric form ( M , 240000) and removal of nucleotides from the tetrameric enzyme causes its dissociation, mainly into the enzymatically active dimeric form and partly into the inactive monomeric form [7]. Formation of the monomer is greater when the dimeric and tetrameric forms are treated at low temperature [7]. Nucleotides, various acyl-CoA derivatives, CoASH, polyols and a high concentration of salt afford partial or complete protection of the enzyme against inactivation or dissociation by cold [8]....

Effect ofin vitrodigested cod liver oil of different quality on oxidative, proteomic and inflammatory responses in the yeastSaccharomyces cerevisiaeand human monocyte-derived dendritic cells

“…Later its role was re‐characterised as mainly being active for the CoA transfer from succinyl‐CoA to acetate, implying an acetate detoxification, and only a minor acetyl‐CoA‐hydrolase activity . Furthermore, ROS induced oxidative inactivation of acetyl‐CoA hydrolase, purified from the supernatant fraction of rat liver, was reported by Nakanishi et al Orlandi et al provided evidence in yeast that ACH1 inactivation severely impairs mitochondrial functions, which influences the acetate metabolism, the chronological lifespan and the occurrence of apoptosis. Additionally, Eisenberg et al , showed that deletion of the ACH1 caused cytosolic accumulation of the acetyl‐CoA precursor acetate, which led to hyperactivation of nucleocytosolic acetyl‐CoA‐synthetase (Acs2p), responsible for conversion of acetate to acetyl‐CoA.…”

Inhibition of Desulfovibrio gigas hydrogenase with copper salts and other metal ions

“…In other cases the inhibition by copper takes place only in the presence of reductants like ascorbate. Some examples are acetylcholinesterase [23], phosphoglucomutase [29], acetyl-CoA hydrolase [30] and catalase [31]. The deleterious effect of the axorbate/ Cu(11) mixture is usually observed only in the presence of oxygen since the mechanism of inactivation involves oxygen radicals generated in a Fenton-type reaction [32].…”

Molecular cloning and functional expression of rat liver cytosolic acetyl‐CoA hydrolase

“…Because of its extreme cold lability, this enzyme had to be purified at room temperature [1–5,9–15], which caused a great loss of the enzyme activity, presumably due to denaturation of the enzyme protein by oxidation and degradation by contaminating proteases. Recently, we found that Triton X‐100, a stabilizer for the membranous proteins and a cytosolic phospholipase A 2 [16], stabilizes CACH also (unpublished data).…”