FUNCTION OF CONSERVED HISTIDINE-RESIDUES IN MAMMALIAN DIHYDROOROTASE

Dihydroorotase (DHOase, EC 3.5.2.3) catalyzes the reversible cyclization of carbamyl aspartate to form dihydroorotate, the third step in de novo pyrimidine biosynthesis. In mammals this activity is carried by the zinc-containing domain of the 243 kDa multifunctional protein CAD. We have replaced conserved residues in the cloned 46 kDa DHOase domain by site-directed mutagenesis. Mutants His1471Ala and His1473Ala lacked catalytic activity, judging by their failure to complement a DHOase-deficient Escherichia coli strain, and were unable to coordinate the active site zinc ion in zinc blotting experiments. This result confirmed earlier predictions. A mutant protein in which the third suspected zinc ligand was changed, Glu1512Asn, had a k(cat) similar to that of the intact CAD molecule and a K-m similar to that of the wild-type recombinant DHOase, observations that argue against a role for glutamate 1512 in catalysis. Mutant His1590Asn had no measurable catalytic activity. This histidine residue was tentatively identified as the third zinc ligand by the failure of the mutant to bind the full complement of zinc in atomic absorption measurements. Mutant His1690Asn had a k(cat) 34-fold lower and a K-m 9-fold higher than those of wild-type recombinant. The kinetic parameters of the mutant His1642Asn were also altered, but to a lesser extent. Diethyl pyrocarbonate (DEPC) was shown previously to inactivate mammalian DHOase. Spectroscopic studies and [C-14]DEPC incorporation demonstrated that the loss of activity is associated with the modification of approximately two histidine residues located at or near the active site. Atomic absorption showed that reaction with DEPC did not result in expulsion of the zinc ion, suggesting that histidine residues 1471, 1473, and 1590 do not react with DEPC. Moreover, incorporation of [C-14]DEPC into mutants His1590Asn and His1642Asn was similar to that observed for the wild-type protein. In contrast, the incorporation of DEPC into the mutant His1690Asn was reduced, and substrate no longer affected the reaction. Furthermore, the strong pH dependence of the catalytic reaction was significantly decreased in this mutant. Our results suggest that the active site zinc is coordinated by three histidines, His1471, His1473, and His1590, and that His1690 is located at the active site and participates in catalysis.