Interaction of substrates and inhibitors with the human fatty acid synthase

Mammalian cells contain a type I fatty acid synthase (FASI) in which all enzyme activities are encoded by a single polypeptide chain. Under normal dietary conditions, FASI is downregulated. However, the overexpression of FASI in tumor cells has led to the hypothesis that this enzyme might be a valid target for anticancer drug discovery. Initially, expression of the human FASI (HFAS) was attempted in E. coli with various pET vectors and induction conditions. But, the production of soluble protein was unattainable due to inclusion body production. Alternatively, using the baculovirus expression system the HFAS has been overexpressed and purified. Triclosan, a common antibacterial additive in personal care products, has an IC 50 of 50 muM for the enoyl reductase component of FASI. In an attempt to identify more potent FASI inhibitors, a number of triclosan analogs have been screened; however, triclosan remains the most active diphenyl ether so far identified. In order to direct the synthesis of novel FASI inhibitors, a detailed enzymological analysis of FASI was undertaken. kcat and Km values were determined for overall and for each partial FAS activity. The role of Lys-1699 (K1699) in cofactor binding by enoyl reductase was explored. Modification of this lysine by pyridoxal phosphate was previously reported to only impact enoyl reductase activity. In agreement with this observation, the K1699A and K1699Q FASI mutants showed a large increase in the Km of enoyl reductase for NADPH. Interestingly, the NADPH Km of the beta-ketoacyl reductase activity was also increased, suggesting that the two NADPH binding sites communicate. In addition, to investigate whether discrepancies between the kcat value for the overall FASI reaction and that observed for the enoyl reductase component result from adventitious loading of the endogenous ACP by exogenously added substrates, a S2151A mutant was also prepared. This serine is the point of attachment of the phosphopantetheine to the FASI ACP; however, replacement of this residue with alanine results in loss of all partial activities, suggesting that the phosphopantetheine moiety plays a structural role in the protein.