Substrate Recognition by the Human Fatty-acid Synthase

Abstract

The human fatty-acid synthase (HFAS) is a potential target for anti-tumor drug discovery. As a prelude to the design of compounds that target the enoyl reductase (ER) component of HFAS, the recognition of NADPH and exogenous substrates by the ER active site has been investigated. Previous studies demonstrate that modification of Lys-1699 by pyridoxal 5-phosphate results in a specific decrease in ER activity. For the overall HFAS reaction, the K1699A and K1699Q mutations reduced k cat and k cat /K NADPH by 8-and 600-fold, respectively (where K NADPH indicates the K m value for NADPH). Thus, Lys-1699 contributes 4 kcal/mol to stabilization of the rate-limiting transition state following NADPH binding, while also stabilizing the most stable ground state after NADPH binding by 3 kcal/mol. A similar effect of the mutations on the ER partial reaction was observed, in agreement with the proposal that Lys-1699 is located in the ER NADPH-binding site. Most unexpectedly, however, both k cat and k cat /K NADPH for the ␤-ketoacyl reductase (BKR) reaction were also impacted by the Lys-1699 mutations, raising the possibility that the ER and BKR activities share a single active site. However, based on previous data indicating that the two reductase activities utilize distinct cofactor binding sites, mutagenesis of Lys-1699 is hypothesized to modulate BKR activity via allosteric effects between the ER and BKR NADPH sites.Fatty acid synthesis generates important intermediates for the construction of cell membranes and for energy storage (1). In humans the reactions resulting in the production of fatty acids are catalyzed by a multifunctional enzyme complex (HFAS), 3 consisting of the following seven catalytic activities: acetyl/malonyl transacylase, ␤-ketoacyl synthase, ␤-ketoacyl reductase (BKR), ␤-hydroxyacyl dehydratase (DH), enoyl reductase (ER), and thioesterase (2-10). In addition, there is an acyl carrier protein (ACP) domain, which carries fatty acyl intermediates in the form of fatty acyl thioesters (5). Palmitate, a C 16 -saturated fatty acid, is the major product produced (5, 11). Fatty acid synthesis, although important evolutionarily for survival during famines (1), was thought to have very little significance for today's modern society (1,9,10,12). This is because of the dietary intake of lipids, which causes HFAS to be down-regulated in normal cells (6,7,13,14). However, HFAS overexpression in many cancers, such as in breast, prostate, and colon tumors, and also in many pre-malignant growths, has led to the hypothesis that HFAS is an important target for the study of tumor biology (7,13,14).Our laboratory has a long standing interest in the development of antibacterial compounds that target prokaryotic fatty acid synthesis. Thus, an initial interest in HFAS was as a control system for validating the specificity of our bacterial FAS inhibitors. However, as described above, HFAS is now thought to be a bona fide target in its own right as a putative anti-tumor target. There have also been suggestions that HFAS inhibi...

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