Substrate Binding and Reactivity are Not Linked: Grafting a Proton-Transfer Network into a Class 1A Dihydroorotate Dehydrogenase

摘要

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 Class 1A mutant 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 mutation of Leu61Phe tightens dihydrooroate binding, but weakens the binding of benzoate inhibitors of Class 1A enzymes.