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

We report that the K12G mutation in triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in (1) a similar to 50-fold increase in K-m for the substrate glyceraldehyde 3-phosphate (GAP) and a 60-fold increase in K-i for competitive inhibition by the intermediate analogue 2-phosphoglycolate, resulting from the loss of stabilizing ground state interactions between the alkylammonium side chain of Lys-12 and the ligand phosphodianion group; (2) a 12000-fold decrease in k(cat) for isomerization of GAP, suggesting a tightening of interactions between the side chain of Lys-12 and the substrate on proceeding from the Michaelis complex to the transition state; and (3) a 6 x 10(5)-fold decrease in k(cat)/K-m, corresponding to a total 7.8 kcal/mol stabilization of the transition state by the cationic side chain of Lys-12. The yields of the four products of the K12G TIM-catalyzed isomerization of GAP in D2O were quantified as dihydroxyacetone phosphate (D H A P) (27%), [1(R)-H-2]DHAP (23%), [2(R)-H-2]GAP (31%), and methylglyoxal (18%) from an enzyme-catalyzed elimination reaction. The K12G mutation has only a small effect on the relative yields of the three products of the transfer of a proton to the TIM-bound enediol(ate) intermediate in D2O, but it strongly favors catalysis of the elimination reaction to give methylglyoxal. The K12G mutation also results in a >= 14-fold decrease in k(cat)/K-m for isomerization of bound glycolaldehyde (GA), although the dominant observed product of the mutant enzyme-catalyzed reaction of [1-C-13]GA in D2O is [1-C-13,2,2-di-H-2]GA from a nonspecific protein-catalyzed reaction. The observation that the K12G mutation results in a large decrease in k(cat)/K-m for the reactions of both GAP and the neutral truncated substrate [1-C-13]GA provides evidence for a stabilizing interaction between the cationic side chain of Lys-12 and the negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate piece.