Catalysis by hamster dihydroorotase: zinc binding, site-directed mutagenesis, and interaction with inhibitors.
Hamster dihydroorotase is the central domain of a trifunctional protein 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 enabled identification of three ligands of zinc at the catalytic site as His15, 17 and 158. The underlined amino acids of the nonapeptide sequence Ile12-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-->ZnII form a triad at the active site and that Arg19, for which even the conservative mutation Arg19-->Lys yields 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 65Zn2+. The Km for dihydroorotate (DHO) increased from 4.0 to 11 microM, while the Vmax decreased from 1.2 to 0.53 mumol 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 Km and a 16-fold decrease in Vmax, indicating involvement of this conserved residue in both binding and catalysis. The mutation Lys239-->Gly increased the Km for DHO 110-fold with a 2-fold increase in Vmax, suggesting that this residue may form a hydrogen bond with the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Williams
N K
NK
Department of Biochemistry, University of Sydney, Australia.
Manthey
M K
MK
Hambley
T W
TW
O'Donoghue
S I
SI
Keegan
M
M
Chapman
B E
BE
Christopherson
R I
RI
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D006224:Cricetinae; D004080:Dihydroorotase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D004952:Esters; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism*
D000596:Amino Acids; D004791:Enzyme Inhibitors; D004952:Esters; D009838:Oligodeoxyribonucleotides; D004080:Dihydroorotase; D015032:Zinc