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

Identification and Characterization of Mitochondrial Acetyl-Coenzyme A Hydrolase from Pisum sativum L. Seedlings

“…As incorporation of labeled acetate into lipids by isolated pea chloroplast is about one-fifth of the maximal rates of acetyl-CoA synthetase, the observed acetylCoA hydrolase activity would be sufficient to provide the necessary acetate for acetyl-CoA synthetase and fatty acid synthesis. Moreover, Budde et al (unpublished data) measured acetyl-CoA levels in active respiring mitochondria which ranged between 150 and 300 ,M. As these values are similar to the Km measured for acetyl-CoA hydrolase (74-150 ,uM; Table III; [9]) and well above the Km for citrate synthase (1 1-15 ,M; [7]) suggests that there would be sufficient substrate for acetyl-CoA hydrolase to provide acetate for fatty acid synthesis. These results are consistent with the compartmentation scheme formulated by Stumpf and coworkers (13,19) in spinach leaves and extends this observation to pea leaf tissue.…”

“…These two factors along with the short incubation times used by Givan and Hodgson (7) would prevent (in the absence of OAA) enough accumulation of CoA-SH to be detected by the DTNB assay. As a result, the low acetyl-CoA and protein concentrations in their reaction mixture favored the more active citrate synthase (7). The pea mitochondrial acetyl-CoA hydrolase is quite sensitive to product inhibition by CoA-SH.…”

“…Citrate synthetase's higher affinity for acetyl-CoA [Km (acetyl-CoA) = The flux through citrate synthase will be primarily controlled by the levels of OAA since the OAA concentration in plant mitochondria (<1 gM; [5]) is less than the Km value for OAA measured for citrate synthase (1-5 pm; [7]). The intramitochondrial OAA concentration is largely determined by the malate concentration and the NADH/NAD+ ratio because of the unfavorable equilibrium of the malate dehydrogenase reaction (5 (16).…”

“…Liedvogel and Stumpf (13), using a radioisotope assay, measured an average specific activity of 47 nmol/h/mg protein for spinach mitochondrial acetyl-CoA hydrolase. At these rates and the protein concentration used by Givan and Hodgson (7), an incubation time of greater than 30 min was required to detect acetyl-CoA hydrolytic activity. Givan and Hodgson (7) incubated the mitochondrial extract no longer than 10 min.…”

“…The acetate formed would diffuse out of the mitochondria and reesterify with CoA-SH by chloroplastic acetyl-CoA synthetase. Givan and Hodgson (7) questioned the general presence of this compartmentation scheme in plants when they were unable to detect acetyl-CoA hydrolytic activity in pea mitochondria. The only acetyl-CoA hydrolysis they observed was OAA dependent and attributed to citrate synthase.…”