| Abstract
| - To obtain a clearer understanding of the forces involved in transition state stabilization byEscherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substratebinding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that theaction of cytidine deaminase is not diffusion-limited. Thus, Km appears to be a true dissociation constant,and kcat describes the chemical reaction of the ES complex, not product release. Enzyme−substrateassociation is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the EScomplex proceeds to the transition state (ES⧧), there is little further change in entropy, but heat is takenup that almost matches the heat that was released with ES formation. As a result, kcat/Km (describing theoverall conversion of the free substrate to ES⧧) is almost invariant with changing temperature. The freeenergy barrier for the enzyme-catalyzed reaction (kcat/Km) is much lower than that for the spontaneousreaction (knon) (ΔΔG⧧ = −21.8 kcal/mol at 25 °C). This difference, which also describes the virtual bindingaffinity of the enzyme for the activated substrate in the transition state (S⧧), is almost entirely enthalpicin origin (ΔΔH = −20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize theES⧧ complex. Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasingtemperature. When a hydrogen bond between Glu-91 and the 3‘-hydroxyl moiety of cytidine is disruptedby truncation of either group, kcat/Km and transition state affinity are each reduced by a factor of 104. Thiseffect of mutation is entirely enthalpic in origin (ΔΔH ∼ 7.9 kcal/mol), somewhat offset by a favorablechange in the entropy of transition state binding. This increase in entropy is attributed to a loss of constraintson the relative motions of the activated substrate within the ES⧧ complex. In an Appendix, some objectionsto the conventional scheme for transition state binding are discussed.
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