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 alpha-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 kcat/Km = 0.26 M-1 s-1. Exogenous phosphite dianion results in a very large increase in the observed second-order rate constant (kcat/Km)obsd for turnover of glycolaldehyde, and the dependence of (kcat/Km)obsd on [HPO32-] exhibits saturation. The data give kcat/Km = 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 (Kd = 38 mM) is 700-fold weaker than its binding to the fleeting complex of TIM with the altered substrate in the transition state (Kd = 53 muM); 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 alpha-carbonyl carbon.
Amyes
Tina L
TL
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, New York 14260-3000, USA.
Richard
John P
JP
D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D000838:Anions / Q000378:metabolism; D017666:Deuterium Oxide / Q000378:metabolism; D004789:Enzyme Activation; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism*; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D017905:Phosphites / Q000494:pharmacology*; D011817:Rabbits; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000378:metabolism*
D000838:Anions; D017905:Phosphites; D005986:Glyceraldehyde 3-Phosphate; D014305:Triose-Phosphate Isomerase; D000079:Acetaldehyde; D017666:Deuterium Oxide; C010972:glycolaldehyde