Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris

Abstract

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.

Fatty acid synthesis in mitochondria of Euglena gracilis

“…smegmatis [18, 291. It is also noted that the pyridine nucleotide requirement of the Euglenu mitochondrial system is different from those of the fatty-acid-synthetic systems in cytosol and chloroplasts of the same organism, which require NADPH for maximum activity [7,101. It has been demonstrated that E. gracilis cells gain ATP by the synthesis of wax esters from the reserve polysaccharide in anoxia, namely wax ester fermentation [I].…”

“…The reaction mixture contained in 0.3 ml, 0.5 mM [l-'4C]acetyl-CoA (0.05 pCi), 2mM NADH, 1 mM MgCI,, I00mM Hepes-KOH buffer, pH 7.0, and enzyme solution. When subcellular distribution of fatty acid synthesis from acetyl-CoA was determined NADPH was also added at 2 mM in the reaction mixture, since NADPH was reported to be required for maximum activity in cytosolic and microsomal fatty acid synthesis [7,111. The reaction was initiated by the addition of enzyme solution and conducted at 30 C for 1 h under aerobic or anaerobic conditions.…”

The Mitochondrion of Euglena gracilis

“…The first two systems, localized in the chloroplasts, are acyl-carrier protein dependent systems (FAS II), which synthesize products with chain lengths of C16 and C18 that can be further elongated to C20-C24 (Ernst-Fonberg and Bloch 1971;Hendren and Bloch 1980). The third, a multifunctional fatty acid synthase (FAS I) located in the cytosol of Euglena, has as main products C16 fatty acids with C14 and as minor products C18 chain lengths (Goldberg and Bloch 1972;Walker et al 1981). The fourth is a microsomal fatty acid synthase activity (Khan and Kolattukudy 1973).…”

Mitochondrial trans-2-Enoyl-CoA Reductase of Wax Ester Fermentation from Euglena gracilis Defines a New Family of Enzymes Involved in Lipid Synthesis

“…The first two are acyl carrier proteindependent systems (FAS II) 1 localized in the chloroplasts and synthesize products with chain lengths of C16 and C18 that can be further elongated to C20 -C24 (10,11). The third, a multifunctional fatty acid synthase (FAS I) whose main products are C16 fatty acids with C14 and C18 chain lengths as minor products, is located in the cytosol of Euglena (12,13). The fourth is a microsomal fatty acid synthase activity (14).…”