Role of Lys-12 in Catalysis by Triosephosphate Isomerase: A Two-Part Substrate Approach
- 18 May 2010
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 49 (25), 5377-5389
- https://doi.org/10.1021/bi100538b
Abstract
We report that the K12G mutation in triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in (1) a ∼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 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 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; and (3) a 6 × 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 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 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 the negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate “piece”.Keywords
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