Isolation of rat liver microsomal short-chain β-ketoacyl-coenzyme A reductase and trans-2-enoyl-coenzyme A hydratase: Evidence for more than one hydratase

Abstract

An enzyme preparation (IIIB) isolated from liver microsomes of untreated male rats was found to contain two activities—short-chain trans-2-enoyl-CoA hydratase and β-ketoacyl-CoA reductase. The hydratase was purified more than 1000-fold, while the reductase activity was purified over 600-fold. Employing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a single band with a molecular weight of 76,000 was observed. Although attempts to separate these two activities have failed, it remains to be established whether the final preparation contains a single enzyme with two activities or two separate enzymes. The hydratase was most active toward crotonyl-CoA, followed by trans-2-hexenoyl-CoA (6:1) and -octenoyl-CoA (8:1); the enzyme was essentially inactive toward substrates containing more than eight carbon atoms. The V max for crotonyl-CoA was 2117 μmol/min/mg protein, while the K m was 59 μ m. Using acetoacetyl-CoA as substrate, the V max for the β-ketoacyl-CoA reductase was over 60 μmol/min/mg protein and the K m was 37μ m; the V max for β-ketopalmitoyl-CoA was only 15% of that observed with acetoacetyl-CoA, although the K m was 6 μ m. During the course of purification, a second short-chain hydratase was discovered (fraction IVA); unlike IIIB, this fraction catalyzed the hydration of 4:1, 6:1, and 8:1 at similar rates. The partially purified preparation yielded maximal activity with 8:1 CoA (apparent V max 35 μmol/min/mg), followed by 6:1 CoA, 4:1 CoA, and 10:1 CoA; longer chain CoA's were relatively poor substrates, with trans-2-hexadecenoyl CoA about 0.1 as active as 8:1 CoA. On SDS-gels, fraction IVA contained four bands, all of which were below 60,000 M r. Proteases, such as trypsin, chymotrypsin, and subtilisin, were found to completely inactivate both enzyme fractions.