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 kcat similar to that of the intact CAD molecule and a Km 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 kcat 34-fold lower and a Km 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 [14C]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.(ABSTRACT TRUNCATED AT 250 WORDS)
Zimmermann
B H
BH
Department of Biochemistry, University of Puerto Rico, San Juan 00936-5067.
Kemling
N M
NM
Evans
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006224:Cricetinae; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004080:Dihydroorotase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D006639:Histidine; D004080:Dihydroorotase; D015032:Zinc; D004047:Diethyl Pyrocarbonate