We report that the K12G mutation at triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in: (1) A ca. 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 analog 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 12,000-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. (3) A 6 × 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 D 2 O were quantified as: dihydroxyacetone phosphate (DHAP), 27%; [1(R)-2 H]-DHAP, 23%; [2(R)-2 H]-GAP, 31%; and 18% methylglyoxal from an enzyme-catalyzed elimination reaction. The K12G mutation has only a small effect on the relative yields of the three products of proton transfer to the TIM-bound enediol(ate) intermediate in D 2 O, 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 [1-13 C]-GA in D 2 O is [1-13 C, 2,2-di-2 H]-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 [1-13 C]-GA provides evidence for a stabilizing interaction between the cationic side chain of Lys-12 and negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate "piece".Triosephosphate isomerase (TIM) 1 catalyzes the stereospecific, reversible, 1,2-hydrogen shift at dihydroxyacetone phosphate (DHAP) to give (R)-glyceraldehyde 3-phosphate (GAP) by a single-base (Glu-165) proton transfer mechanism through an enzyme-bound cisenediol(ate) intermediate (Scheme 1) (1,2). The enzyme's low molecular weight (dimer, 26 kDa/subunit), high cellular abundance (3), and the centrality of proton transfer at carbon in metabolic processes (4-6) have made TIM a prominent target for studies on the mechanism of enzyme action (1,7-10).