Stoichiometry of substrate binding to rat liver fatty acid synthetase

Abstract

Two rat liver fatty acid synthetase preparations, containing 1.6 and 2.0 mol of 4'-phosphopantetheine/mol of synthetase, showed specific activity of 2006 and 2140 nmol of NADPH oxidized/min per mg of protein respectively. The two synthetase preparations could be loaded with either 3.3-4.4 mol of [1-14] acetate or 2.9-3.7 mol of [2-14C]malonate, by incubation with either [1-14C] acetyl-CoA or [2-14C]malonyl-CoA. The 4'-phosphopantetheine site could be more than 90% saturated and the serine site about 80% saturated with malonate derived from malonyl-CoA. However, with acetyl-CoA as substrate, binding at both the 4'-phosphopantetheine and cysteine thiol sites did not reach saturation. We interpret these results to indicate that, whereas the equilibrium constant for transfer of substrates between the serine loading site and the 4'-phosphopantetheine site is close to unity, that for transfer of acetyl moieties between the 4'-phosphopantetheine and cysteine sites favours formation of the 4'-phosphopantetheine thioester. Thus, despite the apparent sub-stoichiometric binding of acetate, the results are consistent with a functionally symmetrical model for the fatty acid synthetase which permits simultaneous substrate binding at two separate active centres.

Amino acid sequences of substrate-binding sites in chicken liver fatty acid synthase

“…Previous results have suggested that the acetyl group is not extensively bound to cysteine (Yuan & Hammes, 1985). Mikkelsen et al (1985c) have reported that ~15% of the acetyl groups are on cysteine for the rat liver fatty acid synthase. The maximum number of acetyl groups bound per enzyme molecule is about 4 [cf.…”

Fatty acid synthase, a proficient multifunctional enzyme

“…Cleavage of the synthase by other proteases made it possible to assign locations to the other component activities on the subunit protein. Domain I contains the NH2 terminus of the polypeptide, the active cysteine-SH of the 3-ketoacyl synthase, and the common serine-OH of the acetyl and malonyl transacylases (Joshi et al, 1970;Stoops & Wakil, 1982;Tsukamoto et al, 1983;Mikkelsen et al, 1985b). Hence, this domain functions as a site for entry of the substrates, the acetyl and malonyl groups, and their subsequent condensation to form carbon-carbon bonds (Figure 1).…”

“…Earlier studies suggested that the acetyl and malonyl groups are bound to the synthase via thioester linkages (Joshi et al, 1970;Philips et al, 1970;Stoops & Wakil, 1982;Mikkelsen et al, 1985b). The acetyl group is bound to the cysteine-SH of the 3-ketoacyl synthase and to the cysteamine-SH of the 4,-phosphopantetheine.…”

“…The malonyl group binds only to the cysteamine-SH of the prosthetic group, 4'-phosphopantetheine; hence, it is presumed to be channeled directly from the active serine O-ester to the ACP-pantetheine-SH (Figure 3) (Joshi et al, 1970;Phillips et al, 1970). The acetyl group, on the other hand, binds to both the active cysteine-SH of the /3-ketoacyl synthase and to the cysteamine-SH of ACP (Joshi et al, 1970;Mikkelsen et al, 1985b). It is not clear, therefore, whether the acetyl group is transferred directly from the serine O-ester to the cysteine-SH or whether it is first transferred to the cysteamine-SH and then to the cysteine-SH of the condensing domain.…”

A Method of Immobilization on the Solid Support of Complex and Simple Enzymes Retaining Their Activity

“…The amino or the carboxylic groups of the peptides can often be employed for coupling to the matrix (10,11). Another group of methods involves coupling multi-subunit enzymes to a Sepharose 4B matrix containing covalently attached specific antibodies (12) or to a metal-chelating resin via a histidine tag linked to the protein (13). Different types of solid support have also been described (13,14).…”