Aspartate Transcarbamylase: Kinetic studies of the catalytic subunit

Abstract

Abstract 1. Steady state kinetics indicates that the binding of substrates by the catalytic subunit of aspartate transcarbamylase is ordered. Product inhibition patterns show that carbamyl phosphate binds first, aspartate binds second, carbamylaspartate dissociates first, and phosphate dissociates second. 2. Nonlinear product inhibition and substrate inhibition indicate that several dead end complexes are formed: aspartate binds to the enzyme-phosphate complex, a 2nd mole of phosphate binds to the enzyme-phosphate complex, a 2nd mole of carbamyl phosphate binds to the enzyme-carbamyl phosphate complex, and carbamylaspartate binds weakly to the free enzyme. 3. The interaction of the catalytic subunit with analogues of both substrates has been studied in an attempt to reveal those structural features of the substrates that are required for binding and function. Acetyl phosphate and N-methylcarbamyl phosphate are the only analogues of carbamyl phosphate tested which are substrates, with maximum velocities at pH 7.8 of 2.4% and 0.03% of the maximum velocity with carbamyl phosphate. Since acetyl phosphate lacks the NH2 group of carbamyl phosphate, this group cannot be essential for function. N,N-Dimethylcarbamyl phosphate is not a substrate, but it is a competitive inhibitor, as is any analogue of carbamyl phosphate with a phosphate or phosphonate dianion, suggesting that at least part of the site for carbamyl phosphate is readily accessible. In fact, with the catalytic subunit, even cytidine triphosphate is a competitive inhibitor. 4. Several dicarboxylic acids were found to be competitive inhibitors of l-aspartate. Succinate and maleate are the strongest inhibitors, malonate and d- and l-malate are good inhibitors, whereas fumarate, glutarate, and d- and l-bromosuccinate are poor inhibitors, and d-aspartate does not inhibit significantly. 5. The pH dependence of the dissociation constant for succinate indicates that a group with pKa 7.1 is required to be positively charged for this inhibitor to bind to the enzyme carbamyl phosphate complex. In contrast, the dissociation constants for carbamyl phosphate and phosphonacetamide vary little between pH 6 and pH 9. 6. Product inhibition by carbamylaspartate when aspartate is varied indicates that the constant for dissociation of aspartate from the central complex is much larger than the dissociation constants for unreactive aspartate analogues such as succinate. This indicates that some of the binding energy of aspartate may be used to facilitate the reaction with carbamyl phosphate. 7. The catalytic subunit is unstable at concentrations below 1 µg per ml. There is an immediate decrease in specific activity with decreasing enzyme concentration and a slower irreversible inactivation during incubation at low concentrations. Both the immediate and the slow losses of activity are prevented by the presence of bovine serum albumin (50 µg per ml) in dilute enzyme solutions. However, because bovine serum albumin forms a complex with the catalytic subunit and alters the apparent kinetic parameters, it was not used in these kinetic studies. 8. At pH 7.8 and 28°, the dissociation constant for carbamyl phosphate is 1.4 x 10-5 m, the Km for aspartate is 0.020 m, and the Vmax is 9.0 x 104 moles per min per 100,000 g of subunit. The dissociation constant for carbamyl phosphate and Km for aspartate vary little between pH 6.4 and pH 8.7, whereas Vmax changes greatly, with an optimum near pH 8. With the use of acetyl phosphate instead of carbamyl phosphate, Km for aspartate is unchanged at pH 7.8, but the dissociation constant for acetyl phosphate is 35-fold higher. With N-methylcarbamyl phosphate, Km for aspartate is 7-fold higher and the dissociation constant for N-methylcarbamyl phosphate is 4-fold higher than for carbamyl phosphate.