Mechanism for Activation of Triosephosphate Isomerase by Phosphite Dianion: The Role of a Ligand-Driven Conformational Change

摘要: The L232A mutation in triosephosphate isomerase (TIM) from Trypanosoma brucei results a small 6-fold decrease k(cat)/K(m) for the reversible enzyme-catalyzed isomerization of glyceraldehyde 3-phosphate to give dihydroxyacetone phosphate. In contrast, this leads 17-fold increase second-order rate constant TIM-catalyzed proton transfer reaction truncated substrate piece [1-(13)C]glycolaldehyde ([1-(13)C]-GA) D(2)O, 25-fold third-order pieces GA and phosphite dianion (HPO(3)(2-)), 16-fold K(d) binding HPO(3)(2-) free enzyme. Most significantly, also an 11-fold extent activation enzyme toward turnover by bound HPO(3)(2-). data provide striking evidence that ca. 1.7 kcal/mol stabilization catalytically active loop-closed form TIM (E(c)) relative inactive open (E(o)). We propose is due relief, mutant TIM, unfavorable steric interactions between bulky hydrophobic side chain Leu-232 basic carboxylate Glu-167, catalytic base, which destabilize E(c) E(o).