Enzymatic conformational fluctuations along the reaction coordinate of cytidine deaminase.

Analysis of the crystal structures for cytidine deaminase complexed with substrate analog 3-deazacytidine, transition-state analog zebularine 3,4-hydrate, and product uridine establishes significant changes in the magnitude of atomic-scale fluctuations along the (approximate) reaction coordinate of this enzyme. Differences in fluctuations between the substrate analog complex, transition-state analog complex, and product complex are monitored via changes in corresponding crystallographic temperature factors. Previously, we reported that active-site conformational disorder is substantially reduced in the transition-state complex relative to the two ground-state complexes. Here, this result is statistically corroborated by crystallographic data for fluorinated zebularine 3,4-hydrate, a second transition-state analog, and by multiple regression analysis. Multiple regression explains 70% of the total temperature factor variation through a predictive model for the average B-value of an amino acid as a function of the catalytic state of the enzyme (substrate, transition state, product) and five other physical and structural descriptors. Furthermore, correlations of atomic fluctuation magnitudes throughout the body of each complex are quantified through an auto-correlation function. The transition-state analog complex shows the greatest correlations between temperature factor magnitudes for spatially separated atoms, underscoring the strong ability of this reaction-coordinate species to "organize" enzymatic fluctuations. The catalytic significance for decreased atomic-scale motions in the transition state is discussed. A thermodynamic argument indicates that the significant decreases in local enzymatic conformational entropy at the transition state result in enhanced energetic stabilization there.