CATALYTIC STRATEGY OF CITRATE SYNTHASE - SUBUNIT INTERACTIONS REVEALED AS A CONSEQUENCE OF A SINGLE AMINO-ACID CHANGE IN THE OXALOACETATE BINDING-SITE

The active site of pig heart citrate synthase contains a histidine res idue (H320) which interacts with the carbonyl oxygen of oxaloacetate a nd is implicated in substrate activation through carbonyl bond polariz ation, a major catalytic strategy of the enzyme. We report here the ef fects on the catalytic mechanism of changing this important residue to glycine. H320G shows modest impairment in substrate Michaelis constan ts [(7-16)-fold] and a large decrease in catalysis (600-fold). For the native enzyme, the chemical intermediate, citryl-CoA, is both hydroly zed and converted back to reactants, oxaloacetate and acetyl-CoA. In t he mutant, citryl-CoA is only hydrolyzed, indicating a major defect in the condensation reaction. As monitored by the carbonyl carbon's chem ical shift, the extent of oxaloacetate carbonyl polarization is decrea sed in all binary and ternary complexes. As indicated by the lack of r apid H320G-oxaloacetate catalysis of the exchange of the methyl proton s of acetyl-CoA or the pro-S-methylene proton of propionyl-CoA, the ac tivation of acetyl-CoA is also faulty. Reflecting this defect in acety l-CoA activation, the carboxyl chemical shift of H320G-bound carboxyme thyl-CoA (a transition-state analog of the neutral enol intermediate) fails to decrease on formation of the H320G-oxaloacetate-carboxymethyl -CoA ternary complex. Progress curves and steady-state data with H320G using citryl-CoA as substrate show unusual properties: substrate inhi bition and accelerating progress curves. Either one of two models with subunit cooperativity [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88; Koshland, D. E., Jr., Nemethy, G., & Filmer, D. (1966) Biochemistry 5, 365] quantitatively accounts for both the init ial velocity data and the individual progress curves. The concentratio ns of all enzyme forms and complexes are assumed to rapidly reach thei r equilibrium Values compared to the rate of substrate turnover. The n ative enzyme also behaves according to models for subunit cooperativit y with citryl-CoA as substrate. However, the rates of formation/dissoc iation and reaction of complexes are kinetically significant. Comparis ons of the values of kinetic constants between the native and mutants enzymes lead us to conclude that the mutant less readily undergoes a c onformation change required for efficient activation of substrates.