Role of glutamate-104 in generating a transition state analogue inhibitor at the active site of cytidine deaminase

The F-19-NMR resonance of 5-[F-19]Fluoropyrimidin-2-one ribonucleoside moves upfield when it is bound by wild-type cytidine deaminase from Escherichia coli, in agreement with UV and X-ray spectroscopic indications that this inhibitor is bound as the rare 3,4-hydrated species 5-fluoro-3,4-dihydrouridine, a transition state analogue inhibitor resembling an intermediate in direct water attack on 5-fluorocytidine. Comparison of pK(A) values of model compounds indicates that the equilibrium constant for 3,4-hydration of this inhibitor in free solution is 3.5 x 10(-4) M, so that the corrected dissociation constant of 5-fluoro-3,4-dihydrouridine from the wild-type enzyme is 3.9 x 10(-11) M. Very different behavior is observed for a mutant enzyme in which alanine replaces Glu-104 at the active site, and k(cat) has been reduced by a factor of 10(8). 5-[F-19]Fluoropyrimidin-2-one ribnucleoside is strongly fluoresce nt, making it possible to observe that the mutant enzyme binds this inhibitor even more tightly (K-d = 4.4 x 10(-8) M) than does the native enzyme (K-d = 1.1 X 10(-7) M). F-19-NMR indicates, however, Ihat the E104A mutant enzyme binds the inhibitor without modification, in a form that resembles the substrate in the ground state. These results are consistent with a major role for Glu-104, not only in stabilizing the ES double dagger complex in the transition state, but also in destabilizing the ES complex in the ground state.