Homocitrate synthase (acetyl-coenzyme A: 2-ketoglutarate C-transferase; E.C. 2.3.3.14) (HCS) catalyzes the condensation of acetyl-CoA (AcCoA) and alpha-ketoglutarate (alpha-KG) to give homocitrate and CoA. Although the structure of an HCS has not been solved, the structure of isopropylmalate synthase (IPMS), a homologue, has been solved (Koon, N., Squire, C. J., and Baker, E. N. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 8295-8300). Three active site residues in IPMS, Glu-218, His-379, and Tyr-410, were proposed as candidates for catalytic residues involved in deprotonation of the methyl group of AcCoA prior to the Claisen condensation to give homocitrylCoA. All three of the active site residues in IPMS are conserved in the HCS from Saccharomyces cerevisiae. Site-directed mutagenesis has been carried out to probe the role of the homologous residues, Glu-155, His-309, and Tyr-320, in the S. cerevisiae HCS. No detectable activity was observed for the H309A and H309N mutant enzyme, but a slight increase in activity was observed for H309A in the presence of 300 mM imidazole, which is still 1000-fold lower than that of wild type (wt). The E155Q and E155A mutant enzymes exhibited 1000-fold lower activity than wt. The activity of E155A, but not of E155Q, could be partially rescued by formate; a K act of 60 mM with a modest 4-fold maximum activation was observed. In the presence of formate, E155A gives k cat, K AcCoA, and K alpha-KG values of 0.0031 s (-1), 13 muM, and 39 microM, respectively, while a primary kinetic deuterium isotope effect of about 1.4 was obtained on V, with deuterium in the methyl of AcCoA. The pH dependence of k cat for E155A in the presence of formate gave a p K a of 7.9 for a group that must be protonated for optimum activity, similar to that observed for the wt enzyme. However, a partial change was observed on the acid side of the profile, compared to the all or none change observed for wt giving a p K a of about 6.7. The k cat for E155Q decreased at high pH, similar to the wt enzyme, but was pH independent at low pH. The Y320F mutant enzyme only lost 25-fold activity compared to that of the wt, giving k cat, K AcCoA, and K alpha-KG values of 0.039 s (-1), 33 microM, and 140 microM, respectively, and a primary kinetic deuterium isotope effect of 1.3 and 1.8 on V/ K AcCoA and V, respectively; the pH dependence of k cat was similar to that of the wt. These data, combined with a constant pH molecular dynamics simulation study, suggest that a catalytic dyad comprising Glu-155 and His-309 acts to deprotonate the methyl group of AcCoA, while Tyr320 is likely not directly involved in catalysis, but may aid in orienting the reactant and/or the catalytic dyad.