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 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 10(7)-fold. Experiments with highly enriched [(14)C]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 x 10(7) M(-1) s(-1) for OMP to less than 2.5 x 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 x 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 x 10(8) M, representing one of the largest connectivity effects that has been reported for an enzyme reaction.
Miller
B G
BG
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Snider
M J
MJ
Short
S A
SA
Wolfenden
R
R
D001667:Binding, Competitive; D002384:Catalysis; D003653:Decarboxylation; D004926:Escherichia coli; D016297:Mutagenesis, Site-Directed; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity; D014542:Uridine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
C005417:orotidylic acid; D014542:Uridine Monophosphate; D009964:Orotidine-5'-Phosphate Decarboxylase