Product distributions for the reaction of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-13C]-GA) catalyzed by triosephosphate isomerase (TIM) in D2O at pD 7.0 in the presence of phosphite dianion and in its absence were determined by 1H NMR spectroscopy. We observe three products for the relatively fast phosphite-activated reaction (Amyes, T. L., and Richard, J. P. (2007) Biochemistry 46, 5841-5854): [2-13C]-GA from isomerization with intramolecular transfer of hydrogen (12% of products), [2-13C, 2-2H]-GA from isomerization with incorporation of deuterium from D2O at C-2 (64% of the products), and [1-13C, 2-2H]-GA from incorporation of deuterium from D2O at C-2 (23% of products). The much slower unactivated reaction in the absence of phosphite results in formation of the same three products along with the doubly deuterated product [1-13C, 2,2-di-2H]-GA. The two isomerization products ([2-13C]-GA and [2-13C, 2-2H]-GA) are formed in the same relative yields in both the unactivated and the phosphite-activated reactions. However, the additional [1-13C, 2-2H]-GA and the doubly deuterated [1-13C, 2,2-di-2H]-GA formed in the unactivated TIM-catalyzed reaction are proposed to result from a nonspecific reaction(s) at the protein surface. The data provide evidence that phosphite dianion affects the rate, but not the product distribution, of the TIM-catalyzed reaction of [1-13C]-GA at the enzyme active site. They are consistent with the conclusion that both reactions occur at an unstable loop-closed form of TIM, and that activation of the isomerization reaction by phosphite dianion results from utilization of the intrinsic binding energy of phosphite dianion to stabilize the active loop-closed enzyme.

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).

Product yields for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D2O at pD 7.9 catalyzed by wildtype triosephosphate isomerase from Trypanosoma brucei brucei (Tbb TIM) and a monomeric variant (monoTIM) of this wildtype enzyme were determined by 1H NMR spectroscopy, and were compared with the yields determined in earlier work for the reactions catalyzed by TIM from rabbit and chicken muscle [O’Donoghue, A. C, Amyes, T. L. and Richard J.P. (2005), Biochemistry 44, 2610–2621]. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen, d-DHAP from isomerization with incorporation of deuterium from D2O into C-1 of DHAP, and d-GAP from incorporation of deuterium from D2O into C-2 of GAP. The yield of DHAP formed by intramolecular transfer of hydrogen decreases from 49% for the muscle enzymes to 40% for wildtype Tbb TIM to 34% for monoTIM. There is no significant difference in the ratio of the yields of d-DHAP and d-GAP for wildtype TIM from muscle sources and Trypanosoma brucei brucei, but partitioning of the enediolate intermediate of the monoTIM reaction to form d-DHAP is less favorable ((kC1)D/(kC2)D = 1.1) than for the wildtype enzyme ((kC1)D/(kC2)D = 1.7). Product yields for the wildtype Tbb TIM and monoTIM-catalyzed reactions of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-13C]-GA) at pD 7.0 in the presence of phosphite dianion and in its absence were determined by 1H NMR spectroscopy [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry 48, 5769–5778]. There is no detectable difference in the yields of the products of wildtype muscle and Tbb TIM-catalyzed reactions of [1-13C]-GA in D2O. The kinetic parameters for phosphite dianion activation of the reactions of [1-13C]-GA catalyzed by wildtype Tbb TIM are similar to those reported for the enzyme from rabbit muscle TIM [Amyes, T. L., and Richard, J. P. (2007), Biochemistry 46, 5841–5854], but there is no detectable dianion activation of the reaction catalyzed by monoTIM. The engineered disruption of subunit contacts at monoTIM causes movement of the essential side chains of Lys-13 and His-95 away from the catalytic active positions. We suggest that this places an increased demand that the intrinsic binding energy of phosphite dianion be utilized to drive the change in the conformation of monoTIM back to the active structure for wildtype TIM, with the result that there is insufficient binding energy remaining to give a detectable stabilization of the transition state for the monoTIM-catalyzed reaction of [1-13C]-GA.

The K12G mutation at yeast triosephosphate isomerase (TIM) results in a 5.5 × 10 5 -fold decrease in k cat /K m for isomerization of glyceraldehyde 3-phosphate, and the activity of this mutant can be successfully "rescued" by NH 4 + and primary alkylammonium cations. The transition state for the K12G mutant TIM-catalyzed reaction is stabilized by 1.5 kcal/mol by interaction with NH 4 + . The larger 3.9 kcal/mol stabilization by CH 3 CH 2 CH 2 CH 2 NH 3 + is due to hydrophobic interactions between the mutant enzyme and the butyl side chain of the cation activator. There is no significant transfer of a proton from alkylammonium cations to GAP at the transition state for the K12G mutant TIM-catalyzed reaction, because activation by a series of RNH 3 + shows little or no dependence on the pK a of RNH 3 + . A comparison of k cat /K m = 6.6 × 10 6 M −1 s −1 for the wildtype TIM-catalyzed isomerization of GAP and the third-order rate constant of 150 M −2 s −1 for activation by NH 4 + of the K12G mutant TIM-catalyzed isomerization shows that stabilization of the bound transition state by the effectively intramolecular interaction of the cationic side chain of Lys-12 at wildtype TIM is 6.3 kcal/mol greater than for the corresponding intermolecular interaction of NH 4 + at K12G mutant TIM.