Evidence for Two Distinct CoA Binding Sites on Yeast cy- Isopropylmalate Synthase*

cu-Isopropylmalate synthase (EC 4.1.3.12) from Saccharomyces cerevisiae was purified to a purity of about 95%. The molecular weight of the enzyme is approximately 127,000, as determined by sedimentation equilibrium centrifugation and by intersubunit cross-linking. Under denaturing conditions, one major species (95%) with molecular weight of about 65,000 is obtained. The dimeric structure of the enzyme is apparently unaffected by the presence of various ligands, including substrates, the feedback inhibitor leutine, and the inactivating combination of CoA plus Zn”+. Our previous observation that CoA, a product of the reaction, causes a very specific, Zn2+-dependent, reversible inactivation of yeast a-isopropylmalate synthase (Tracy, J., and Kohlhaw, G. B. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1802-1806) was further analyzed. Evidence is presented for the existence of two distinct CoA sites on each enzyme subunit. The first site (product site) interacts with CoA and desulfo-CoA, both of which are competitive inhibitors with respect to acetyl-CoA, with apparent K, values of 70 and 90 PM, respectively. Equilibrium dialysis experiments show that up to one product site per subunit can be saturated with CoA, and that the binding capacity changes in parallel with the specific activity of the enzyme. The dissociation constant for CoA binding at the product site is approximately 65 FM. The second site (“regulatory site”) appears to be absolutely specific for CoA. Binding of CoA to this site occurs only when Zn2+ is present, is independent of the specific activity of the enzyme, and does not eliminate CoA binding at the product site. Thus, when Zn2+ is present, the number of CoA sites saturable per subunit increases by 1.0. A dissociation constant of about 35 FM can be calculated for the Zn2+-dependent binding of CoA. The evolutionary establishment of a second CoA binding site is interpreted as further corroborating the idea that the CoA-mediated inactivation of ru-isopropylmalate synthase has regulatory significance.