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 dihydropyrimidina se from the liver of pig and calf. The V/K for 5,6-dihydrouracil (DHU) (or the alternative substrate glutarimide) measured with either the n ative zinc or cadmium-substituted enzymes decreases at both low and hi gh pH giving pK values of about 7.5-8 and 9-10. The low pK value obser ved in V is perturbed significantly to lower pH (approximately 6), and the high pK is not observed. The binding of glutarate monoamide is op timum when the group with a pK of 7.7 is protonated, and this same gro up must be protonated for the reverse reaction, that is, formation of DHU from N-carbamoyl-beta-alanine. These data are consistent with a ge neral base mechanism and in addition suggest that the enzyme is presen t initially with a water bound to the active site zinc. The enzymic ge neral base with a pK of 7.5-8 is required to activate water for nucleo philic attack on the C-4 of 5,6-dihydrouracil which is directly coordi nated to the active site zinc. The second group with a pK of 9-10 like ly 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 ef fects are near unity on the V/K for the natural substrate 5,6-dihydrou racil, but a finite effect of 1.6 is observed on V. Data suggest a rat e-determining transition state under V/K conditions for which no proto ns 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 dep rotonation of the resulting carboxylic acid product and its subsequent release.