Role of Lys-12 in Catalysis by Triosephosphate Isomerase: A Two-Part Substrate Approach

摘要

We report that the K12G mutation in triosephosphate isomerase (TIM) from Saccharomycesncerevisiae results in (1) a ∼50-fold increase in Km for the substrate glyceraldehyde 3-phosphate (GAP) and an60-fold increase in Ki for competitive inhibition by the intermediate analogue 2-phosphoglycolate, resultingnfrom the loss of stabilizing ground state interactions between the alkylammonium side chain of Lys-12 andnthe ligand phosphodianion group; (2) a 12000-fold decrease in kcat for isomerization of GAP, suggesting antightening of interactions between the side chain of Lys-12 and the substrate on proceeding fromtheMichaelisncomplex to the transition state; and (3) a 6u0001105n-fold decrease in kcat/Km, corresponding to a total 7.8 kcal/molnstabilization of the transition state by the cationic side chain of Lys-12. The yields of the four products of thenK12GTIM-catalyzed isomerization ofGAP inD2Owere quantified as dihydroxyacetone phosphate (DHAP)n(27%), [1(R)-n2nH]DHAP (23%), [2(R)-n2nH]GAP (31%), and methylglyoxal (18%) from an enzyme-catalyzednelimination reaction. TheK12G mutation has only a small effect on the relative yields of the three products ofnthe transfer of a proton to the TIM-bound enediol(ate) intermediate inD2O, but it strongly favors catalysis ofnthe elimination reaction to give methylglyoxal. The K12G mutation also results in a 14-fold decrease in kcat/Kmnfor isomerization of bound glycolaldehyde (GA), although the dominant observed product of the mutantnenzyme-catalyzed reaction of [1-n13nC]GA in D2O is [1-n13nC,2,2-di-n2nH]GA from a nonspecific protein-catalyzednreaction. The observation that the K12G mutation results in a large decrease in kcat/Km for the reactions ofnboth GAP and the neutral truncated substrate [1-n13nC]GA provides evidence for a stabilizing interactionnbetween the cationic side chain of Lys-12 and the negative charge that develops at the enolate-like oxygen innthe transition state for deprotonation of the sugar substrate “piece”.