title: Substrate binding and reactivity are not linked: grafting a proton-transfer network into a Class 1A dihydroorotate dehydrogenase.
abstract: Adding the two residues comprising the conserved proton-transfer network of Class 2 dihydroorotate dehydrogenase (DHOD) to the Cys130Ser Class 1A DHOD did not restore the function of the active site base or rapid flavin reduction. Studies of triple, double, and single mutant Class 1A enzymes showed that the network actually prevents cysteine from acting as a base and that the network residues act independently. Our data show that residue 71 is an important determinant of ligand binding specificity. The Leu71Phe mutation tightens dihydrooroate binding but weakens the binding of benzoate inhibitors of Class 1A enzymes.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0606, United States.
authors.forename: Claudia A
authors.initials: CA
authors.lastname: McDonald
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Palfey
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D000090722:Dihydroorotate Dehydrogenase; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D000090722:Dihydroorotate Dehydrogenase; D011522:Protons; C004768:4,5-dihydroorotic acid; D009963:Orotic Acid; D044925:Oxidoreductases Acting on CH-CH Group Donors