Structural Mutations That Probe the Interactions between the Catalytic and Dianion Activation Sites of Triosephosphate Isomerase

Abstract

Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form D-glyceraldehyde 3-phosphate. The effects of two structural mutations at TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50-fold and 80-fold decreases, respectively, in (kcat/Km)E and (kcat/Km)E•HPi for deprotonation of GA catalyzed by free TIM and by the TIM•HPO32− complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy, or on the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG for 208-YGGS. The L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E•HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy, and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation, but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provide support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst.

Enzyme Architecture: The Effect of Replacement and Deletion Mutations of Loop 6 on Catalysis by Triosephosphate Isomerase

“…aUnder standard assay conditions: 30 mM TEA buffer (pH 7.5) at I = 0.1 (NaCl) and 25 °C.bData from ref (53). …”

“…16,17,33,53,71 Catalysis by the L6RM provides a remarkable example of the plasticity of the structure of c TIM. We find that the severe distortion in the structure of the unliganded wild-type enzyme (Figure 6C) may be overcome by the utilization of phosphodianion binding energy to mold c TIM into the catalytically active closed form.…”

Enzyme architecture: on the importance of being in a protein cage

“…(A) A model for the activation of TIM by HPO 3 2− [3,21,22]. TIM is shown to exist mainly in an inactive open form E O , which is converted to an active high-energy closed form E C ( K C << 1) that is stabilized by interactions with HPO 3 2− .…”

“…Interactions between TIM and the phosphodianion of GAP account for 80% of the enzymatic rate acceleration [19,20], while the binding of phosphite dianion to TIM results in a 4 kcal/mol stabilization of the transition state for the unactivated reaction of glycolaldehyde (GA) [18]. This is not due to a direct stabilizing interaction between the transition state and dianion, because no such interactions are possible [21]. Rather, a large fraction of the intrinsic binding energy of the substrate dianion, or phosphite dianion, is used to drive the energetically demanding change in enzyme conformation, from an inactive open form (E O ,Figure 2A) to the high-energy enzyme cage ( E C , K C << 1), which shows a high reactivity towards deprotonation of GA [3,22].…”

Connecting Active‐Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme

“…Given that residue 100 is aL ys in PfTIM and itss ide chain points away from the F96 aromatic ring, it is tempting to speculate that the absence of additional stabilization of F96 "trans"c onformation is the reasonf or the observation of an intermediate "eclipsed"s tate of the F96 side chain (Table S5). Furthermore, becauseo ft he proximity of residue 96 (S/F) and the hinge residue 167 (V/I/L) side chains, the conformationalt ransitions of residue96a ppear to be coupled to the conformational change of residue 167 (PfTIM PDB ID:1 LYXa nd 1O5X, Figures1Ba nd S5 E, Table S5; Mycobacterium tuberculosis TIM-PDB ID:3TAO, Figure S5 The transition of the apo state to the catalytically competent state is accompanied by the following structuralc hanges (Tables3 andS 6):1 )loop-6 closure and synchronized movement of loop-7 residues that help to anchor the substrate phosphate group (Figure S6), [12,15,16] 2) ac hange in the conformation of loop-6 N-terminal (P166-W168;T able S6) and C-terminal hinge residues (K174A176) andm ovement of the E165 www.chembiochem.org side chain from a" swung-out" (pointing away from active site) to a" swung-in" (pointing into the active site) conformation, and 3) ar esidue 96 side chain flip from "gauche"( c 1 = 468)t o "trans"(c 1 = 1758;F igure S2 and Ta ble 3). These structuralc hanges close the substrate entry/exit route, place the substrate in proximity to the catalytic residues ( Figure S6 Aa nd B) and stabilize the loop-6 "closed"c onformation for the duration of the catalytic cycle.…”