Mutation of amino acids thought to polarize the oxaloacetate carbonyl in citrate synthase severely reduces but does not abolish activity of the enzyme

Oligonucleotide-directed mutagenesis has been used to alter two active site residues of Escherichia coli citrate synthase, histidine-305 and arginine-314. Both residues are thought to be involved in the polarization of the carbonyl group of oxaloacetate and thus facilitate attack at the carbonyl carbon by acetyl-CoA. In one mutant, designated CS305 H→A , His-305 was mutated to alanine and in the other, designated CS314 R→L , Arg-314 was changed to leucine. Both mutants have greatly reduced turnover numbers, less than 0.1% of the wild-type value. The dissociation constant for formation of the binary enzyme–oxaloacetate complex, K i, OAA , is at least 950 μM for CS305 H→A , and about 500 μM for CS314 R→L , 28 and 15 times the wild-type value, respectively. The Michaelis constants for the two substrates, K OAA and K AcCoA , which measure the affinity of the enzyme for the catalytically significant ternary complex, are less radically altered: values of K AcCoA are actually 3.5-fold and 4.6-fold lower for CS305 H→A and CS314 R→L , respectively. These kinetic effects are taken to mean that both His-305 and Arg-314 are important for the successful formation of an efficient transition state, very likely by polarizing the carbonyl group of oxaloacetate as has been suggested, and that the residual kinetic activity, in both mutants, occurs by a mechanism which benefits from only part of this polarization. Allosteric properties of the mutant enzymes, as measured by NADH inhibition and binding, and KCl activation, are normal. Inhibition of the enzyme by α-ketoglutarate, an oxaloacetate analogue, is only slightly weakened by the mutations, a finding which suggests that this analogue is not a substrate itself because it cannot bind in the active site in such a way as to undergo carbonyl polarization.