Transient kinetic studies of fatty acid synthetase. A kinetic self-editing mechanism for the loading of acetyl and malonyl residues and the role of coenzyme A.
A kinetic self-editing mechanism for correcting errors in the loading of thioester substrates is described for the animal fatty acid synthetase reaction. In the catalyzed reaction, these substrates load competitively on a common phosphopantetheine site, and during each of the eight loading steps the enzyme sites are partitioned between competent and incompetent substrate molecules. The incompetently bound substrate is removed by CoA through reversal of the loading reaction and partitioning again occurs. The loading-unloading cycle is repeated until competent enzyme complex is formed and the reaction proceeds. Furthermore, at each step the loading of a malonyl residue is competitively favored as is the unloading of enzyme-bound acetyl groups. This mechanism is entirely consistent with the recently postulated role (Stern, A., Sedgwick, B., and Smith, S. J. Biol. Chem. (1982) 257, 799-803) of CoA as a co-substrate. Supporting evidence is obtained by monitoring the progress curves of NADPH oxidation by chicken liver fatty acid synthetase in the stopped flow apparatus. At noninhibiting acetyl-CoA, the reaction shows an initial lag period as the result of preferential formation of malonyl-enzyme and time-dependent recycling of the loading step to obtain competent acetyl-enzyme. At a malonyl-CoA/acetyl-CoA ratio of 2:1, the induction time of the reaction is 1.02 +/- 0.05 s at 6 degrees C. It decreases with increasing acetyl-CoA concentration or preincubation of the enzyme with acetyl-CoA which promotes acetyl-enzyme formation but is slightly increased upon preincubation with malonyl-CoA. Increasing acetyl-CoA causes a parallel decrease in steady state cycle time (i.e. the average time required to complete a single malonyl-CoA condensation cycle), suggesting that the latter is limited by the lag period. At inhibitory acetyl-CoA, the steady state cycle time is lengthened due to acetyl-enzyme formation at malonyl-CoA loading steps and to the recycling necessary to obtain competent malonyl-enzyme. A requirement of CoA for the first condensation cycle is unequivocally demonstrated in conventional spectrophometric assays and stopped flow experiments by using phosphotransacetylase and acetyl phosphate as a CoA trap. This requirement at each loading step is normally met by CoA generated through initial loading. At noninhibitory acetyl-CoA, added CoA inhibits the reaction and slightly increases the lag.(ABSTRACT TRUNCATED AT 400 WORDS)