Role of enzyme-ribofuranosyl contacts in the ground state and transition state for orotidine 5 '-phosphate decarboxylase: A role for substrate destabilization?

The crystal structure of yeast orotidine 5'-monophosphate decarboxylase (OD Case) complexed with the inhibitor 6-hydroxyuridine 5'-phosphate (BMP) reve als the presence of a series of strong interactions between enzyme residues and functional groups of this ligand. Enzyme contacts with the phosphoribo furanosyl moiety of orotidine 5'-phosphate (OMP) have been shown to contrib ute at least 16.6 kcal/mol of intrinsic binding free energy to the stabiliz ation of the transition state for the reaction catalyzed by yeast ODCase. I n addition to these enzyme-ligand contacts, active site residues contribute d by both subunits of the dimeric enzyme are positioned to form hydrogen bo nds with the 2'- and 3'-OH groups of the ligand's ribosyl moiety. These inv olve Thr-100 of one subunit and Asp-37 of the opposite subunit, respectivel y. To evaluate the contributions of these ribofuranosyl contacts to ground state and transition slate stabilization, Thr-100 and Asp-37 were each muta ted to alanine. Elimination of the enzyme's capacity to contact individual ribosyl OH groups reduced the k(cat)/K-m value of the T100A enzyme by 60-fo ld and that of the D37A enzyme by 300-fold. Removal of the 2'-OH group from the substrate OMP decreased the binding affinity by less than a factor of 10, but decreased k(cat) by more that 2 orders of magnitude. Upon removal o f the complementary hydroxymethyl group from the enzyme, little further red uction in k(cat)/K-m for 2'-deoxyOMP was observed. To assess the contributi on made by contacts involving both ribosyl hydroxyl groups at once, the abi lity of the D37A mutant enzyme to decarboxylate 2'-deoxyOMP was measured. T he value of k(cat)/K-m for this enzyme-substrate pair was 170 M-1 s(-1), re presenting a decrease of more than 7.6 kcal/mol of binding free energy in t he transition state. To the extent that electrostatic repulsion in the grou nd state can be tested by these simple alterations, the results do not lend obvious support to the view that electrostatic destabilization in the grou nd state enzyme-substrate complex plays a major role in catalysis.