CATALYSIS BY HAMSTER DIHYDROOROTASE - ZINC-BINDING, SITE-DIRECTED MUTAGENESIS, AND INTERACTION WITH INHIBITORS

Hamster dihydroorotase is the central domain of a trifunctional protei n which has been cloned, overexpressed, and purified from Escherichia coli. Using the cDNA encoding the dihydroorotase domain, site-directed mutagenesis of amino acid residues conserved between species has enab led identification of three ligands of zinc at the catalytic site as H is15, 17 and 158. The underlined amino acids of the nonapeptide sequen ce 12-Asp13-Val14-His15-Val16-His17-Leu18-Arg19-Glu20 from hamster are conserved between dihydroorotases from 8 species. It is proposed that the residues Asp13-His15-->Zn-II form a triad at the active site and that Arg19, for which even the conservative mutation Arg19-->Lys yield s an inactive enzyme, is involved in substrate binding. Site-directed mutagenesis of the conserved His186-->Ala yielded a mutant enzyme with a reduced affinity for Zn-65(2+) The K-m for dihydroorotate (DHO) inc reased from 4.0 to 11 mu M, while the V-max decreased from 1.2 to 0.53 mu mol min(-1) (mg of protein)(-1), implicating this residue in only a minor way with binding of DHO and in catalysis. The mutation Asp230- ->Glu resulted in a 14-fold increase in K-m and a 16-fold decrease in Vmax, indicating involvement of this conserved residue in both binding and catalysis. The mutation Lys239-->Gly increased the K-m for DHO 11 0-fold with a 2-fold increase in V-max, suggesting that this residue m ay form a hydrogen bond with the substrate. The three-dimensional stru ctures of the methyl esters of the substrates N-carbamyl-L-aspartate a nd L-DHO, and the inhibitors, L-6-thiodihydroorotate (TDHO), oxo-1,2,3 ,6-tetrahydropyrimidine-4,6-dicarboxylate (HDDP), and trans- and cis-2 -oxohexahydropyrimidine-4,6-dicarboxylate (trans- and cis-HTDP) have a lready been determined by X-ray crystallography. Correlation of these structures with their affinities for dihydroorotase suggests that DHO binds to the active site with the exocyclic 4-carboxylate group in the axial orientation. H-1 NMR spectroscopy indicates that the axial conf ormations of DHO and TDHO predominate in aqueous solution. Aided by mo lecular modeling of the portion of the active site formed by the conse rved nonapeptide, roles for amino acid residues in substrate binding a nd catalysis by dihydroorotase are proposed.