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

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 inhibit...

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Bibliographic Details
Published in:Biochemistry (Easton) 2000-07, Vol.39 (28), p.8113-8118
Main Authors: Miller, Brian G, Snider, Mark J, Short, Steven A, Wolfenden, Richard
Format: Article
Language:English
Subjects:
Binding, Competitive
Catalysis
Decarboxylation
Escherichia coli
Mutagenesis, Site-Directed
Orotidine-5'-Phosphate Decarboxylase - chemistry
Orotidine-5'-Phosphate Decarboxylase - genetics
Orotidine-5'-Phosphate Decarboxylase - metabolism
Protein Conformation
Saccharomyces cerevisiae
Substrate Specificity
Uridine Monophosphate - analogs & derivatives
Uridine Monophosphate - chemistry
Uridine Monophosphate - metabolism
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Summary: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 k cat/K m 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, k cat/K m 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 K i 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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi000818x