ESCHERICHIA COLI CITRATE SYNTHASE . PURIFICATION AND EFFECT OF POTASSIUM ON SOME PROPERTIES

Citrate synthase (EC 4.1.3.7) which catalyzes the condensation of acetyl-coenzyme A with oxaloacetate to form citrate, was purified from Escherichia coli extracts. The purified enzyme was homogeneous as judged by ultracentrifugation. Potassium ion markedly increased the stability of the enzyme and lowered the apparent Km’s for acetylcoenzyme A and oxaloacetate. These effects were not specific for potassium, and other monovalent cations were also effective in the decreasing order NH4+ > Na+ > Li+ = (CH3)4 N+. The enzyme showed normal Michaelis-Menten kinetics (Km for acetyl-coenzyme A, 1.1 × 10-4 m; Km for oxaloacetate, 2.1 × 10-5 m) at 0.1 m K+; while lower substrate affinities and nonlinear kinetics were observed when K+ was in low concentration or absent from the reaction mixture. The pH optimum of the enzyme was 8.0. The molecular weight, estimated by gel filtration, was 280,000 daltons, and was independent of the concentration of potassium in the eluent. A shift in the ultraviolet absorption spectrum was observed when potassium was added to the salt-free enzyme. Thus, the enzyme does not aggregate or dissociate in the absence of potassium, but undergoes an apparent conformational change which significantly alters its substrate affinities and sensitivities to various inhibitors. © 1969, American Chemical Society. All rights reserved.