| In dilute aqueous solution, at 25°C, the trimethylsulfonium ion undergoes nucleophilic attack by dimethylamine at a rate of 1 x 10-8 M-1 sec-1. Under otherwise identical conditions, histamine N-methyl transferase catalyzes a similar alkylation at a rate of ∼106 M-1sec-1 , exceeding the knon value by a factor of 1013 fold. More startling is the catalytic rate enhancement produced by OMP decarboxylase (ODCase) of 1017 fold in the chemical conversion of orotidine 5'-phosphate into uridine 5'-phosphate. Bronsted analysis (betalg = 0.7), reported herein, suggests that there is a significant amount of negative charge already transferred to the incipient carbanion at the transition state for similar decarboxylations that occur spontaneously in water. Significant disagreement exists over the transition state structure that arises at the active site of ODCase. Extensive mutational analyses have shown that the enzyme is exquisitely sensitive to any substitution that involves residues that line the substrate binding pocket. In the present work, we examined interactions in the native ODCase from yeast, by binding affinity and spectroscopic measurements using substrate analogues that bear non-reactive substituents at the position that would ordinarily be occupied by the scissile carboxylate group of OMP (I). ODCase binds tightly to the inhibitor, 6-methylamino-UMP(II), but only when its amino substituent is uncharged. This interaction, studied by competitive inhibition experiments and high field 13C NMR, was interpreted as evidence against the hypothesis that OMP decarboxylase operates by a catalytic mechanism involving anion-anion repulsion. The inhibitory complex formed from 6-cyano UMP(III) and OMP decarboxylase was studied by Raman spectroscopy. Upon binding, the Raman stretching frequency of the inhibitor's CN group undergoes a considerable shift toward lower wave number (from 2240 to 2225). Based on studies with the model compound 6-cyano-1,3 dimethyl uracil and on vibrational calculations, the enzyme-induced change in the nitrite stretching frequency is ascribed to desolvation of the ligand and distortion of the ligand in which the pyrimidine base and nitrile group become non-planar. Comparing the inhibitory activity of three isosteric nucleotides in which an amide substituent takes the place of the substrate carboxylate, we found that ODCase binds with equally high affinity (Ki 1.5 nM) to V & VI, in which the amide heteroatom is a chalcogen. ODCase bound much less tightly to the oxygen derivative (IV) by a factor of 26000 fold. To our knowledge, this is the largest effect of an isosteric substitution ever measured in a biological receptor-ligand system. |
