P.: Enzymatic Catalysis of Proton Transfer at Carbon: Activation of Triosephosphate Isomerase by Phosphite Dianion

ABSTRACT: 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 R-hydroxycarbonyl and oxydianion portions of GAP. TIM catalyzes proton transfer from glycolaldehyde in D 2 O, resulting in deuterium incorporation that can be monitored by 1 H 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 [HPO 3 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 q ) 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 R-carbonyl carbon. Despite the wealth of mechanistic and structural data available for enzyme catalysts of proton transfer at R-carbonyl carbon (1-4), the origins of the rate accelerations effected by these enzymes remain elusive. The principal burden for such enzymes is the very large thermodynamic barrier to the formation of simple enolates in aqueous solution (5-7), but the physical mechanism(s) by which they lower this barrier and the nature of the corresponding transition state stabilization remain topics of current interest. Triosephosphate isomerase (TIM) 1 is the prototypical protein catalyst of proton transfer at R-carbonyl carbon and catalyzes the reversible stereospecific 1,2-shift of the pro-R proton at dihydroxyacetone phosphate (DHAP) to give (R)-glyceraldehyde 3-phosphate (GAP) by a single-base (Glu-165) mechanism through a cis-enediol(ate) intermediate (Scheme 1) (8). The early extensive studies of TIM provided a clear description of the chemical events that occur at the enzyme active site (9, 10), and they show that the enzyme approaches "perfection" in its catalysis of the isomerization of triose phosphates (11). TIM presents a unique opportunity for detailed study of the origin of the enzymatic rate acceleration for proton transfer at carbon for several reasons. (1) TIM catalyzes the aldose-ketose isomerization of small and chemically simple phosphodianion substrates, and it requires no cosubstrates, metal ions, or other cofactors (8-10). (2) The mechanism (9, 10) and free energy profile (11) have been well-defined in extensive chemica