Jahnke K, Podschun B, Schnackerz K D, Kautz J, Cook P F
Theodor-Boveri-Institut fuer Biowissenschaften (Biozentrum), Universitaet Wuerzburg, Germany.
Biochemistry. 1993 May 18;32(19):5160-6. doi: 10.1021/bi00070a027.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
