Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate "anchor"

To obtain a clearer understanding of the forces involved in transition stat e stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate binding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited. Thus, K-m a ppears to be a true dissociation constant, and k(cat) describes the chemica l reaction of the ES complex, not product release. Enzyme-substrate associa tion is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the ES complex proceeds to the transition state (ESdouble dagg er), there is little further change in entropy, but heat is taken up that a lmost matches the heat that was released with ES formation. As a result, k( cat)/K-m (describing the overall conversion of the free substrate to ESdoub le dagger) is almost invariant with changing temperature. The free energy b arrier for the enzyme-catalyzed reaction (k(cat)/K-m) is much lower than th at for the spontaneous reaction (k(non)) (Delta Delta G(double dagger) = -2 1.8 kcal/mol at 25 degrees C). This difference, which also describes the vi rtual binding affinity of the enzyme for the activated substrate in the tra nsition state (S'), is almost entirely enthalpic in origin (Delta Delta H = -20.2 kcal/mol), compatible with the formation of hydrogen bonds that stab ilize the ES' complex. Thus, the transition state affinity of cytidine deam inase increases rapidly with decreasing temperature. When a hydrogen bond b etween Glu-91 and the 3'-hydroxyl moiety of cytidine is disrupted by trunca tion of either group, k(cat)/K-m and transition state affinity are each red uced by a factor of 10(4). This effect of mutation is entirely enthalpic in origin (Delta Delta H similar to 7.9 kcal/mol), somewhat offset by a favor able change in the entropy of transition state binding. This increase in en tropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ESdouble dagger complex. In an Appendix, so me objections to the conventional scheme for transition state binding are d iscussed.