Murine adenosine deaminase (ADA), a 40 kDa enzyme, catalyzes the hydrolytic deamination of adenosine to form inosine, enhancing the rate of the spontaneous reaction by more than than 1011 times the rate of the non-catalyzed reaction. This value is directly related to the enzyme's affinity for the altered substrate in the transition state, Ktx, which is approximately equal to 49 aM. ADA was chosen as a model system to analyze transition state affinity and thermodynamics of activation because of its well-known mechanism and structure. Using spectrophotometric assays, the turnover rate, kcat, and catalytic efficiency, kcat/KM, of ADA were determined between temperatures 15-35 oC. By plotting the thermal dependence of the measured rates and applying the resulting kinetic values to the Arrhenius and Eyring-Polanyi equations, thermodynamic activation parameters for ADA catalysis were established. ADA reduces the free energy barrier for adenosine deamination by 16.2 kcal/mol. (ΔG‡ = 14.6 kcal/mol) greater than half the value of the non catalyzed reaction's (ΔG‡ = 30.8 kcal/mol). The free energy barrier between the ground and transition states of ADA's catalysis is comprised of a large enthalpic component (ΔH‡ = 11.4 kcal/mol) and a smaller entropic component (TΔS‡ = -3.2 kcal/mol). The free energy barrier between the unliganded and transition states, related to kcat/KM, was also measured, and was found to be equal to 9 kcal/mol This free energy barrier is made up of a 1.8 kcal/mol change in enthalpy and a -6.9 kcal/mol change in entropy. The results of this study highlight the high affinity of ADA for the transition state and its utilization of favorable changes in enthalpy and entropy to reduce the free energy barrier of activation. Enzyme-substrate interactions between ADA and adenosine play a role in reducing the free energy barrier by providing interactions that can be used by the enzyme to discriminate the transition state from the ground state. The thermal dependence of catalytic rate constants were determined on a truncated substrate and a variant form of ADA to disrupt interactions that are postulated to play a role in transition state discrimination. One hydrogen bond between ADA and adenosine, specifically Asp-19 and adenosine's 5'-hydroxyl moiety, is disrupted using a truncated variant of adenosine, 5'deoxyadenosine. When this interaction is disrupted, kcat decreases almost 2000-fold, and KM increases over 50-fold. These changes are reflected in a diminished affinity for the transition state, from 49 aM to 17 pM. Upon removal of the 5' hydroxyl group, the activation barrier for deamination increases by 4 kcal/mol compared to the intact substrate.This large change in free energy is caused by a 4.2 kcal/mol increase in enthalpy, consistent with the loss of a hydrogen bond between Asp-19 and adenosine's 5'hydroxyl moiety. When the Asp-19 residue is mutated to alanine, its interactions with adenosine's 5' and 3' hydroxyl moieties are disrupted. The enzyme's affinity for the transition state is decreased, although not as much as the elimination of adenosine's 5' hydroxyl group, to 480 fM. The free energy of activation of D19A catalysis of adenosine deamination appears to be closer to that of the native interaction (ΔΔG‡ = 2 kcal/mol). However, this minor change is comprised of a large unfavorable change in entropy, 9 kcal/mol, and a smaller favorable 6.8 kcal/mol change in enthalpy compared to the native reaction. This study highlights the favorable thermodynamic contributions that distant enzyme-substrate interactions make to discriminate for the transition state in enzyme catalysis.