Evidence that the catalytic and regulatory functions of carbamyl-phosphate synthetase from Escherichia coli are not dependent on oligomer formation

Abstract

Carbamyl-phosphate synthetase from E. coli is an allosteric enzyme which undergoes reversible association reactions in phosphate buffer. The positive allosteric effectors, ornithine, IMP and NH3, facilitate oligomer formation, but UMP, a negative effector, prevents or decreases oligomer formation. When the enzyme is immobilized by reaction with activated Sepharose, under conditions where the enzyme exists only as a monomer, nearly full catalytic activity is retained and the effects of ornithine, IMP and UMP on the catalytic activity as a function of MgATP concentration are not significantly altered. Gel-filtration chromatography on Sephadex G-200 of catalytic quantities of the enzyme in the presence of all substrates showed that the elution volume was the same as that measured for the enzyme under conditions where it is known to exist in the monomer form. The specific activity of the enzyme does not increase when the concentration of the enzyme is increased 100-fold from a concentration at which the enzyme exists as monomer to a level at which the enzyme exists predominantly as oligomer. The monomer form of the enzyme is apparently the principle active species, and oligomer formation is probably not directly related to enzyme activity or enzyme regulation.