title: Mechanism for activation of triosephosphate isomerase by phosphite dianion: the role of a hydrophobic clamp.
abstract: The role of the hydrophobic side chains of Ile-172 and Leu-232 in catalysis of the reversible isomerization of R-glyceraldehyde 3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) by triosephosphate isomerase (TIM) from Trypanosoma brucei brucei (Tbb) has been investigated. The I172A and L232A mutations result in 100- and 6-fold decreases in k(cat)/K(m) for the isomerization reaction, respectively. The effect of the mutations on the product distributions for the catalyzed reactions of GAP and of [1-(13)C]-glycolaldehyde ([1-(13)C]-GA) in D(2)O is reported. The 40% yield of DHAP from wild-type Tbb TIM-catalyzed isomerization of GAP with intramolecular transfer of hydrogen is found to decrease to 13% and to 4%, respectively, for the reactions catalyzed by the I172A and L232A mutants. Likewise, the 13% yield of [2-(13)C]-GA from isomerization of [1-(13)C]-GA in D(2)O is found to decrease to 2% and to 1%, respectively, for the reactions catalyzed by the I172A and L232A mutants. The decrease in the yield of the product of intramolecular transfer of hydrogen is consistent with a repositioning of groups at the active site that favors transfer of the substrate-derived hydrogen to the protein or the oxygen anion of the bound intermediate. The I172A and L232A mutations result in (a) a >10-fold decrease (I172A) and a 17-fold increase (L232A) in the second-order rate constant for the TIM-catalyzed reaction of [1-(13)C]-GA in D(2)O, (b) a 170-fold decrease (I172A) and 25-fold increase (L232A) in the third-order rate constant for phosphite dianion (HPO(3)(2-)) activation of the TIM-catalyzed reaction of GA in D(2)O, and (c) a 1.5-fold decrease (I172A) and a larger 16-fold decrease (L232A) in K(d) for activation of TIM by HPO(3)(2-) in D(2)O. The effects of the I172A mutation on the kinetic parameters for the wild-type TIM-catalyzed reactions of the whole substrate and substrate pieces are consistent with a decrease in the basicity of the carboxylate side chain of Glu-167 for the mutant enzyme. The data provide striking evidence that the L232A mutation leads to a ca. 1.7 kcal/mol stabilization of a catalytically active loop-closed form of TIM (E(C)) relative to an inactive open form (E(O)).
authors.affiliation: Department of Chemistry, University at Buffalo, the State University of New York, Buffalo, New York 14260-3000, USA.
authors.forename: M Merced
authors.initials: MM
authors.lastname: Malabanan
authors.forename: Astrid P
authors.initials: AP
authors.lastname: Koudelka
authors.forename: Tina L
authors.initials: TL
authors.lastname: Amyes
authors.forename: John P
authors.initials: JP
authors.lastname: Richard
mesh_terms: D004099:Dihydroxyacetone Phosphate / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D005986:Glyceraldehyde 3-Phosphate / Q000737:chemistry / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007536:Isomerism; D008958:Models, Molecular; D017905:Phosphites / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism
chemical_list: D017905:Phosphites; D005986:Glyceraldehyde 3-Phosphate; D004099:Dihydroxyacetone Phosphate; D014305:Triose-Phosphate Isomerase