Divalent metal derivatives of the hamster dihydroorotase domain.

Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides. The recombinant hamster DHOase domain from the trifunctional protein, CAD, was overexpressed in Escherichia coli and purified. The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6.0. The apoenzyme was reconstituted with different divalent cations at pH 7.4. Co(II)-, Zn(II)-, Mn(II)-, and Cd(II)-substituted DHOases had enzymic activity, but replacement with Ni(2+), Cu(2+), Mg(2+), or Ca(2+) ions did not restore activity. Atomic absorption spectroscopy showed binding of one Co(II), Zn(II), Mn(II), Cd(II), Ni(II), or Cu(II) to the enzyme, while Mg(II) and Ca(II) were not bound. The maximal enzymic activities of the active, reconstituted DHOases were in the following order: Co(II) --> Zn(II) --> Mn(II) --> Cd(II). These metal substitutions had major effects upon values for V(max); effects upon the corresponding K(m) values were less pronounced. The pK(a) values of the Co(II)-, Mn(II)-, and Cd(II)-substituted enzymes derived from pH-rate profiles are similar to that of Zn(II)-DHOase, indicating that the derived pK(a) value of 6.56 obtained for Zn-DHOase is not due to ionization of an enzyme-metal aquo complex, but probably a histidine residue at the active site. The visible spectrum of Co(II)-substituted DHOase exhibits maxima at 520 and 570 nm with molar extinction coefficients of 195 and 210 M(-1) cm(-1), consistent with pentacoordination of Co(II) at the active site. The spectra at high and low pH are different, suggesting that the environment of the metal binding site is different at these pHs where the reverse and forward reactions, respectively, are favored.