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 (FAS), Ser819 (acetyltransferase, OHAC) and Ser5421 (malonyl/palmitoyl transferase, OHMa1) of subunit beta and Ser180 (pantetheine binding site, SHc) and Cys1305 (3-oxoacyl synthase, SHp) of subunit alpha were replaced, by targeted in vitro mutagenesis, by the non-acylatable amino acids glutamine, glycine or alanine. The four mutated FAS proteins together with two pairs of double mutants (OHAc/OHMa1 and SHc/SHp) were episomally expressed in appropriate delta fas1 or delta fas2 deletion strains. The purified enzymes isolated from these transformants were used for comparative acyl binding studies with the substrates [1-14C]acetyl-CoA and [2-14C]malonyl-CoA. Malonate was found to be transacylated to enzyme-bound pantetheine (SHc) exclusively by the Ser5421 hydroxyl group of malonyltransferase (OHMa1) while acetate could use both the acetyl (Ser819) and the malonyl (Ser5421) transferase active sites on its way to the SHc and SHp binding sites. Acylation of SHc with either substrate was unaffected by the absence of the 'peripheral' SH group (SHp) while binding of acetate to SHp was dependent on enzyme-bound pantetheine (SHc). These genetic data support a revised model regarding the intra-molecular channeling of acetate and malonate within yeast fatty acid synthase. Quantitative acyl binding studies revealed a maximum 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 represented performic-acid-labile thioester bonds. The binding characteristics of both substrates, exhibiting Hill coefficients distinctly lower than 1, as well as their non-linear Lineweaver-Burk and Scatchard plots, point to a marked negative cooperativity among the 12 yeast FAS subunits. The observed sub-stoichiometric substrate binding characteristics of the enzyme are ascribed to this effect. An a priori asymmetry of the complex appears unlikely since the coenzyme-A:FAS transacylation equilibrium may be shifted towards the fully acetylated enzyme in the presence of N-ethylmaleimide. In contrast to the limited acylation capacity 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 Km of the coenzyme-A:OHAc transacylation reaction keeps the acyl-O-ester content of the acylated enzyme at less than 5% of the total.(ABSTRACT TRUNCATED AT 250 WORDS)
Schuster
H
H
Lehrstuhl für Biochemie, Universität Erlangen-Nürnberg, Germany.
Rautenstrauss
B
B
Mittag
M
M
Stratmann
D
D
Schweizer
E
E
D000085:Acetates / Q000378:metabolism; D019342:Acetic Acid; D000215:Acylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis; D008314:Malonates / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000085:Acetates; D005227:Fatty Acids; D008314:Malonates; C030290:malonic acid; D064429:Fatty Acid Synthases; D019342:Acetic Acid