Contribution of Enzyme−Phosphoribosyl Contacts to Catalysis by Orotidine 5‘-Phosphate Decarboxylase

Publisher Website
Google Scholar
Abstract
The crystal structure of the complex formed between recombinant yeast orotidine 5‘-phosphate decarboxylase and the competitive inhibitor 6-hydroxyuridine 5‘-phosphate reveals the presence of four hydrogen bonds between active site residues Tyr-217 and Arg-235 and the phosphoryl group of this inhibitor. When Tyr-217 and Arg-235 are individually mutated to alanine, values of kcat/Km are reduced by factors of 3000- and 7300-fold, respectively. In the Y217A/R235A double mutant, activity is reduced more than 107-fold. Experiments with highly enriched [14C]orotic acid show that when ribose 5‘-phosphate is deleted from substrate orotidine 5‘-phosphate, kcat/Km is reduced by more than 12 orders of magnitude, from 6.3 × 107 M-1 s-1 for OMP to less than 2.5 × 10-5 M-1 s-1 for orotic acid. Activity toward orotate is not “rescued” by 1 M inorganic phosphate. The Ki value of ribose 5‘-phosphate, representing the part of the natural substrate that is absent in orotic acid, is 8.1 × 10-5 M. Thus, the effective concentration of the 5‘-phosphoribosyl group, in stabilizing the transition state for enzymatic decarboxylation of OMP, is estimated to be >2 × 108 M, representing one of the largest connectivity effects that has been reported for an enzyme reaction.