Formation of coenzyme A from acetyl coenzyme A in pea leaf mitochondria: A requirement for oxaloacetate and the absence of hydrolysis

Abstract

The release of coenzyme A-SH (CoA-SH) from acetyl CoA was measured in preparations of sonicated mithchondria prepared from Pisum sativum leaves on a discontinous Percoll gradient. Under our assay conditions, we found negligibsle releases of CoA-SH from acetyl CoA in the absence of a o-substrate. Co A-SH formation proceeded rapidly in the presence of oxaloacetate (OAA), indicating that the principal enzyme responsible for CoA-SH release is citrate synthase (EC 4.1.3.7.). OAA was consumed and citrate produced by this reaction. The apparent K m (acetyl CoA) was about 11–15 μM, and the apparent K m (OAA) was about 8–10 μM. We argue that in pea leaf mitochondria the great majority of the acetyl CoA produced by pyruvate oxidation will most likely enter into citrate formation and that the acetyl units will thereby pass into the tricarboxylic-acid cycle. Our data do not indicate any substantial hydrolytic release of CoA-SH from acetyl CoA. We therefore suggest that the acetyl-CoA hydrolase reported in spinach leaf mitochondria is not active in pea leaf mitochondria. Schemes have been proposed which invoke hydrolysis of mitochondrial acetyl CoA as a source of acetate for fatty acid synthesis in chloroplasts, but this type of scheme is evidently not valid for pea leaves.