ACID-BASE CATALYTIC MECHANISM OF DIHYDROPYRIMIDINASE FROM PH STUDIES

The pH dependence of kinetic parameters and solvent deuterium isotope effects have been used to probe the mechanism of the dihydropyrimidinase from the liver of pig and calf. The V/K for 5,6-dihydrouracil (DHU) (or the alternative substrate glutarimide) measured with either the native zinc or cadmium-substituted enzymes decreases at both low and high pH giving pK values of about 7.5-8 and 9-10. The low pK value observed in V is perturbed significantly to lower pH (approximately 6), and the high pK is not observed. The binding of glutarate monoamide is optimum when the group with a pK of 7.7 is protonated, and this same group must be protonated for the reverse reaction, that is, formation of DHU from N-carbamoyl-beta-alanine. These data are consistent with a general base mechanism and in addition suggest that the enzyme is present initially with a water bound to the active site zinc. The enzymic general base with a pK of 7.5-8 is required to activate water for nucleophilic attack on the C-4 of 5,6-dihydrouracil which is directly coordinated to the active site zinc. The second group with a pK of 9-10 likely reflects Zn-water ionization of the free enzyme. The water bound to the active site Zn is displaced by reactant binding, and thus the pK of 9-10 is not observed in the V profile. Solvent deuterium isotope effects are near unity on the V/K for the natural substrate 5,6-dihydrouracil, but a finite effect of 1.6 is observed on V. Data suggest a rate-determining transition state under V/K conditions for which no protons are in flight. Some transition state after hydrolysis of the amide bond for which one or more protons are in flight likely limits under V conditions. The latter solvent-sensitive step likely includes the deprotonation of the resulting carboxylic acid product and its subsequent release.