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 CS305H----A, His-305 was mutated to alanine and in the other, designated CS314R----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, Ki, OAA, is at least 950 microM for CS305H----A, and about 500 microM for CS314R----L, 28 and 15 times the wild-type value, respectively. The Michaelis constants for the two substrates, KOAA and KAcCoA, which measure the affinity of the enzyme for the catalytically significant ternary complex, are less radically altered: values of KAcCoA are actually 3.5-fold and 4.6-fold lower for CS305H----A and CS314R----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.(ABSTRACT TRUNCATED AT 250 WORDS)
Anderson
D H
DH
Department of Chemistry, University of Manitoba, Winnipeg, Canada.
Duckworth
H W
HW
D000596:Amino Acids / Q000235:genetics*; D001665:Binding Sites; D002950:Citrate (si)-Synthase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009154:Mutation; D010071:Oxaloacetates; D007652:Oxo-Acid-Lyases / Q000378:metabolism*
D000596:Amino Acids; D010071:Oxaloacetates; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases