ACTIVE-SITE MUTANTS OF ESCHERICHIA-COLI CITRATE SYNTHASE - EFFECTS OFMUTATIONS ON CATALYTIC AND ALLOSTERIC PROPERTIES

We report properties of five active site mutants of Escherichia coli c itrate synthase, in which histidine 264, aspartate 362, and phenylalan ine 383 were replaced by alanines, and arginines 387 and 407 by leucin es. All mutants have much lower turnover numbers than wild type enzyme ; the strongest effect was with the arginine 387 mutant, perhaps becau se the substrate, oxaloacetate, binds in a different orientation. The arginine 407 mutant has lost most of its ability to distinguish alpha- ketoglutarate, a competitive inhibitor, from oxaloacetate. The mutatio ns of histidine 264 and aspartate 362 affect steady-state kinetics as would be anticipated from current models for citrate synthase catalysi s, and resemble mutations of these residues, in pig heart and E. coli enzyme, reported by others. Mutations of residues 264,362, and 383 als o affect allosteric properties. With the phenylalanine 383 mutant, ace tyl-CoA saturation is strongly sigmoid, even in the presence of the ac tivator, KCl, implying a marked shift of the allosteric equilibrium to ward the T state. The histidine 264 mutant appears to be shifted towar d R state and shows weaker binding of the allosteric inhibitor, NADH; thus this mutation also affects the allosteric site, 25-30 angstrom aw ay.