SUBSTRATE AND PRODUCT BINDING-SITES OF YEAST FATTY-ACID SYNTHASE - STOICHIOMETRY AND BINDING-KINETICS OF WILD-TYPE AND IN-VITRO MUTATED ENZYMES

The four known substrate binding sites of yeast fatty acid synthase (F AS), Ser819 (acetyltransferase, OHAc) and Ser5421 (malonyl/palmitoyl t ransferase, OHMal) of subunit beta and Ser180 (pantetheine binding sit e, SHc) and Cys1305 (3-oxoacyl synthase, SHp) of subunit alpha were re placed, by targeted in vitro mutagenesis, by the non-acylatable amino acids glutamine, glycine or alanine. The four mutated FAS proteins tog ether with two pairs of double mutants (OHAc/OHMal and SHc/SHp) were e pisomally expressed in appropriate Delta fas1 or Delta fas2 deletion s trains. The purified enzymes isolated from these transformants were us ed for comparative acyl binding studies with the substrates [1-C-14]ac etyl-CoA and [2-C-14]malonyl-CoA. Malonate was found to be transacylat ed to enzyme-bound pantetheine (SHc) exclusively by the Ser5421 hydrox yl group of malonyltransferase (OHMal) while acetate could use both th e acetyl (Ser819) and the malonyl (Ser5421) transferase active sites o n its way to the SHc and SHp, binding sites. Acylation of SHc with eit her substrate was unaffected by the absence of the 'peripheral' SH gro up (SHp) while binding of acetate to SHp was dependent on enzyme-bound pantetheine (SHc). These genetic data support a revised model regardi ng the intra-molecular channeling of acetate and malonate within yeast fatty acid synthase. Quantitative acyl binding studies revealed a max imum of 2-3 mol rather than the expected 12 mol of malonate and of 6-7 mol rather than 24 mol of acetate bound/mol hexameric yeast FAS. Only 20-30% of the malonyl-enzyme and 35-50% of the acetyl enzyme represen ted per formic-acid-labile thioester bonds. The binding characteristic s of both substrates, exhibiting Hill coefficients distinctly lower th an 1, as well as their non-linear Lineweaver-Burk and Scatchard plots, point to a marked negative cooperativity among the 12 yeast FAS subun its. The observed sub-stoichiometric substrate binding characteristics of the enzyme are ascribed to this effect. An a priori asymmetry of t he complex appears unlikely since the coenzyme-A:FAS transacylation eq uilibrium may be shifted towards the fully acetylated enzyme in the pr esence of N-ethylmaleimide. In contrast to the Limited acylation capac ity of the 'resting' enzyme, complete acylation of yeast FAS at all of its 12 SHc and SHp sites is observed under steady-state conditions of fatty acid biosynthesis. Under these conditions, the enzyme exhibits full-site reactivity at its SHp, SHc and OHAc sites, but a concomitant 18-fold increase in K-m of the coenzyme-A:OHAc transacylation reactio n keeps the acyl-O-ester content of the acylated enzyme at less than 5 % of the total. Thus, the differential acyl binding characteristics of yeast fatty acid synthase with its strictly limited capacity for acet ate and malonate obviously ensure that essentially the full complement of binding sites is reserved for the intermediates and products of th e reaction process.