Mechanistic Imperatives for Deprotonation of Carbon Catalyzed by Triosephosphate Isomerase: Enzyme-Activation by Phosphite Dianion.

The mechanistic imperatives for catalysis of deprotonation of α-carbonyl carbon by triosephosphate isomerase (TIM) are discussed. There is a strong imperative to reduce the large thermodynamic barrier for deprotonation of carbon to form an enediolate reaction intermediate; and, a strong imperative for specificity in the expression of the intrinsic phosphodianion binding energy at the transition state for the enzyme-catalyzed reaction. Binding energies of 2 and 6 kcal/mol, respectively, have been determined for formation of phosphite dianion complexes to TIM and to the transition state for TIM-catalyzed deprotonation of the truncated substrate glycolaldehyde [T. L. Amyes, J. P. Richard, Biochemistry2007, 46, 5841]. We propose that the phosphite dianion binding energy, which is specifically expressed at the transition state complex, is utilized to stabilize a rare catalytically active loop-closed form of TIM. The results of experiments to probe the role of the side chains of Ile172 and Leu232 in activating the loop-closed form of TIM for catalysis of substrate deprotonation are discussed. Evidence is presented that the hydrophobic side chain of Ile172 assists in activating TIM for catalysis of substrate deprotonation through an enhancement of the basicity of the carboxylate side-chain of Glu167. Our experiments link the two imperatives for TIM-catalyzed deprotonation of carbon by providing evidence that the phosphodianion binding energy is utilized to drive an enzyme conformational change, which results in a reduction in the thermodynamic barrier to deprotonation of the carbon acid substrate at TIM compared with the barrier for deprotonation in water. The effects of a P168A mutation on the kinetic parameters for the reactions of whole and truncated substrates are discussed.