Carbamoyl-phosphate synthetase: an example of effects on enzyme properties of shifting an equilibrium between active monomer and active oligomer

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Abstract
Carbamoyl-phosphate synthetase from Escherichia coli is subject to allosteric activation by ornithine, allosteric inhibition by uridine 5''-phosphate (UMP), and reversible concentration-dependent self-association. Positive allosteric effectors, magnesium adenosine 5''-triphosphate (MgATP), K+, and inorganic phosphate facilitate association. The purpose of this study was to determine the state of association of carbamoyl-phosphate synthetase in the presence and absence of different substrates and effectors and to consider the basis for the observed effects of enzyme concentration on specific activity. Studies employing gel filtration chromatography have shown that when the concentration of carbamoyl-phosphate synthetase is low (< 0.01 mg/mL), the enzyme exists as monomer under all conditions, including the presence of UMP in phosphate buffer and the presence of all substrates plus ornithine (conditions that support maximal catalytic activity). At higher enzyme concentrations (e.g., > 0.01 mg/mL) the specific activity increases with increasing enzyme concentration when MgATP is nonsaturing but is independent of enzyme concentration when MgATP is saturating or when ornithine is present with MgATP being either saturating or nonsaturating. These results indicate that the catalytic activity of this enzyme is not directly linked to oligomer formation. The theoretical properties and possible significance of a generalized model of enzyme association-dissociation in which the active monomeric form, in equilibrium with another monomeric form, is specifically subject to self-association but the different states of association have the same specific activity, are discussed. This generalized model together with the results with carbamoyl-phosphate synthetase suggests that the effects characteristic of associating enzyme systems may arise in some circumstances through processes in which different states of association do not necessarily have different catalytic activities and/or ligand-binding properties.