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 ∼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 - 10⁵-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 (DHAP) (27%), [1(R)-²H]DHAP (23%), [2(R)-²H]GAP (31%), and methylglyoxal (18%) from an enzyme-ca alyzed 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- ¹³C]GA in D₂O is [1-¹³C,2,2-di-²H]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 [1-¹³C]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";.