| Abstract: | An initial activity burst lasting 5 to 10 s is observed for both de novo synthesis with acetyl-CoA as primer and for elongation of palmitoyl-CoA catalyzed by the multienzyme complex fatty acid synthetase from Mycobacterium smegmatis. After the initial burst, synthetase activity slows at least 6-fold to the steady state rate. The size of the initial burst is proportional to the amount of synthetase protein and corresponds to the synthesis of a small number C three to five) of C24 or C26 acyl chains per mol of enzyme. During the initial burst, C24, C26 acyl enzyme is formed and can be isolated by ammonium sulfate precipitation. On incubation with CoA, enzyme-bound acyl chains undergo transacylation to form the corresponding CoA derivatives. Diffusion of C24-CoA and C26-CoA from the enzyme is slow and rate-limiting for overall fatty acid synthesis. Mycobacterial polysaccharides markedly accelerate this rate-determining step but bovine serum albumin does not. This facilitation of product diffusion accounts for the large stimulation of de novo synthesis and of elongation of mycobacterial polysaccharide. It is also shown that the high apparent Km for acetyl-CoA (approximately 400 micrometer) in the steady state reflects the substrate concentration required to shift the product pattern in favor of shorter chain fatty acids (C16,C18). These conditions circumvent the slow, rate-limiting diffusion of C24-CoA and C26-CoA. |
