Enzymatic catalysis of proton transfer at carbon: Activation of triosephosphate isomerase by phosphite dianion

More than 80% of the rate acceleration for enzymatic catalysis of the aldose-ketose isomerization of (R)-glyceraldehyde 3-phosphate (GAP) by triosephosphate isomerase (TIM) can be attributed to the phosphodianion group of GAP [Amyes, T. L., O'Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325-11326]. We examine here the necessity of the covalent connection between the phosphodianion and triose sugar portions of the substrate by "carving up" GAP into the minimal neutral two-carbon sugar glycolaldehyde and phosphite dianion pieces. This "two-part substrate" preserves both the α-hydroxycarbonyl and oxydianion portions of GAP. TIM catalyzes proton transfer from glycolaldehyde in D₂O, resulting in deuterium incorporation that can be monitored by ¹H NMR spectroscopy, with k_cat/K_m = 0.26 M^-1 s^-1. Exogenous phosphite dianion results in a very large increase in the observed second-order rate constant (k_cat\K_m)_obsd for turnover of glycolaldehyde, and the dependence of (k_cat/K _m)_obsd on [HPO₃^2-] exhibits saturation. The data give k_cat/K_m = 185 M^-1 s^-1 for turnover of glycolaldehyde by TIM that is saturated with phosphite dianion so that the separate binding of phosphite dianion to TIM results in a 700-fold acceleration of proton transfer from carbon. The binding of phosphite dianion to the free enzyme (K_d = 38 mM) is 700-fold weaker than its binding to the fleeting complex of TIM with the altered substrate in the transition state (K_d^‡ = 53 μM); the total intrinsic binding energy of phosphite dianion in the transition state is 5.8 kcal/mol. We propose a physical model for catalysis by TIM in which the intrinsic binding energy of the substrate phosphodianion group is utilized to drive closing of the "mobile loop" and a protein conformational change that leads to formation of an active site environment that is optimally organized for stabilization of the transition state for proton transfer from α-carbonyl carbon.