We report that the K12G mutation at triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in: (1) A ca. 50-fold increase in Km for the substrate glyceraldehyde 3-phosphate (GAP) and a 60-fold increase in Ki 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 kcat 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 x 105-fold decrease in kcat/Km, 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 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 D2O, but it strongly favors catalysis of the elimination reaction to give methylglyoxal. The K12G mutation also results in a >/= 14-fold decrease in kcat/Km 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 kcat/Km 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 negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate "piece".
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|