Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain

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Abstract
Palmitoyl-CoA hydrolase (EC 3.1.2.2) catalyzes the irreversible hydrolysis of long-chain acyl-CoA thioesters. This enzyme is found primarily in the postmicrosomal supernatant fraction prepared from homogenates of rat brain. Either of 2 forms of the hydrolase, a lower-MW species of approximately 70,000 or a higher-MW species of approximately 130,000 can be isolated by gel filtration. The higher-MW form is obtained from columns of Sephadex G-200 eluted with buffer containing 10 .mu.M-palmitoyl-CoA or 20% (vol/vol) glycerol, whereas the lower-MW form is obtained when the eluting buffer does not contain palmitoyl-CoA or glycerol. The 2 forms of the hydrolase have the same pH optimum of 7.5, are equally sensitive to the benzoic acid), and exhibit the same Km (1.8 .mu.M) with palmitoyl-CoA as substrate. The 2 forms differ in the availabiliy or reactivity of certain external thiol groups, as determined by covalent chromatography with activated thiol Sepharose. Dilute solutions of the lower-MW form of the hydroxylase rapidly lose activity (50% in 60 min at 0.degree. C), but there is no change in the Km with palmitoyl-CoA as substrate during this progressive inactivation. Dilutions of the hydrolase in buffer containing 10 .mu.M-palmitoyl-CoA retain full activity. However, addition of palmitoyl-CoA to solutions of the lower-MW form will not restore previously lost hydrolase activity. Apparently the substrate palmitoyl-CoA promotes the formation of a relatively stable dimer from 2 unstable subunits. This process may not be reversible, since the removal of palmitoyl-CoA or glycerol from solutions of the higher-MW form does not result in the appearance of the lower-MW form of the hydrolase.