Purification and characterization of beta-ketoacyl-ACP synthetase I from Spinacia oleracea leaves

beta-Ketoacyl-acyl carrier protein (ACP) synthetase I was purified 180-fold from crude extracts of spinach leaves. The purified preparation was completely free from other component enzymes of the de novo fatty acid synthetase (FAS) system. Its molecular weight was estimated to be 56,000 by gel filtration. The apparent Km value for malonyl-CoA in the presence of ACP and malonyl-CoA:ACP transacylase was 4 microM. Purified synthetase I was highly active with acyl-ACP having chain lengths from C2 to C14, with hexanoyl-ACP being the most effective substrate, but palmitoyl-ACP was far less effective and stearoyl-ACP almost inactive. The antibiotic, cerulenin, strongly inhibited synthetase I activity. The inhibition by cerulenin was protected by prior incubation with hexanoyl-ACP, decanoyl-ACP, and myristoyl-ACP. The synthetase was inhibited by 1 mM p-CMB and 5 mM NEM, but not by 1 mM arsenite.