Bg. Miller et al., Contribution of enzyme-phosphoribosyl contacts to catalysis by orotidine 5'-phosphate decarboxylase, BIOCHEM, 39(28), 2000, pp. 8113-8118
The crystal structure of the complex formed between recombinant yeast oroti
dine 5'-phosphate decarboxylase and the competitive inhibitor 6-hydroxyurid
ine 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
or. 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. E
xperiments with highly enriched [C-14]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 o
rotate 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 absen
t in orotic acid, is 8.1 x 10(-5) M. Thus, the effective concentration of t
he 5'-phosphoribosyl group, in stabilizing the transition state for enzymat
ic decarboxylation of OMP, is estimated to be >2 x 10(8) M, representing on
e of the largest connectivity effects that has been reported for an enzyme
reaction.