Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of inactivation by chelators and steady-state kinetic properties

Abstract

Dihydropyrimidine amidohydrolase (EC 3.5.2.2) catalyzes the reversible hydrolysis of 5,6-dihydropyrimidines to the corresponding β-ureido acids. Previous work has shown that incubation of this Zn 2+ metalloenzyme with 2,6-dipicolinic acid, 8-hydroxyquinoline-5-sulfonic acid, or o-phenanthroline results in inactivation by Zn 2+ removal by a reaction pathway involving formation of a ternary enzyme-Zn 2+-chelator complex which subsequently dissociates to yield apoenzyme and the Zn 2+-chelate (K. P. Brooks, E. A. Jones, B. D. Kim, and E. G. Sander, (1983) Arch. Biochem. Biophys. 226,469–483). In the present work, the pH dependence of chelator inactivation is studied. The equilibrium constant for formation of the ternary complex is strongly pH dependent and increases with decreasing pH for all three chelators. There is a positive correlation between the value of the equilibrium constant observed for each chelator and the value of its stability constant for formation of Zn 2+-chelate. The affinity of the chelators for the enzyme increases in the order 8-hydroxyquinoline-5-sulfonic acid > o-phenanthroline > 2,6 dipicolinic acid. The first-order rate constant for breakdown of the ternary complex to yield apoenzyme and Zn 2+-chelate is invariant with pH for a given chelator but is different for each chelator, increasing in the reverse order. The pH dependence of the inactivation shows that two ionizable groups on the enzyme are involved in the inactivation. On the other hand, the steady-state kinetic behavior of the enzyme is welldescribed by ionization of a single group with a p K of 6.0 in the free enzyme. The basic form of the group is required for catalysis; protonation of the group decreases both V max and the apparent affinity for substrate. Conversely, binding of substrate decreases the p K of this group to about 5. l-Dihydroorotic acid is shown to be a competitive inhibitor of dihydropyrimidine amidohydrolase. Binding of l-dihydroorotic acid increases the p K of the ionizable group to 6.5. The agreement between the p K in the enzyme- l-dihydroorotic acid complex and the higher p K observed in the pH dependence of inactivation by chelators suggests that the same group is involved in the binding of substrate, l-dihydroorotic acid, and chelators. The different effects of substrate and l-dihydroorotic acid on the pK suggest that the binding modes of these two ligands may be different and suggest a structural basis for the mutally exclusive substrate specificities of dihydropyrimidine amidohydrolase and dihydroorotase.