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

Abstract

Two mutations of the phosphodianion gripper loop in chicken muscle triosephosphate isomerase (cTIM) were examined: (1) the loop deletion mutant (LDM) formed by removal of residues 170–173 [Pompliano, D. L., et al. (1990) Biochemistry 29, 3186–3194] and (2) the loop 6 replacement mutant (L6RM), in which the N-terminal hinge sequence of TIM from eukaryotes, 166-PXW-168 (X = L or V), is replaced by the sequence from archaea, 166-PPE-168. The X-ray crystal structure of the L6RM shows a large displacement of the side chain of E168 from that for W168 in wild-type cTIM. Solution nuclear magnetic resonance data show that the L6RM results in significant chemical shift changes in loop 6 and surrounding regions, and that the binding of glycerol 3-phosphate (G3P) results in chemical shift changes for nuclei at the active site of the L6RM that are smaller than those of wild-type cTIM. Interactions with loop 6 of the L6RM stabilize the enediolate intermediate toward the elimination reaction catalyzed by the LDM. The LDM and L6RM result in 800000- and 23000-fold decreases, respectively, in kcat/Km for isomerization of GAP. Saturation of the LDM, but not the L6RM, by substrate and inhibitor phosphoglycolate is detected by steady-state kinetic analyses. We propose, on the basis of a comparison of X-ray crystal structures for wild-type TIM and the L6RM, that ligands bind weakly to the L6RM because a large fraction of the ligand binding energy is utilized to overcome destabilizing electrostatic interactions between the side chains of E168 and E129 that are predicted to develop in the loop-closed enzyme. Similar normalized yields of DHAP, d-DHAP, and d-GAP are formed in LDM- and L6RM-catalyzed reactions of GAP in D2O. The smaller normalized 12–13% yield of DHAP and d-DHAP observed for the mutant cTIM-catalyzed reactions compared with the 79% yield of these products for wild-type cTIM suggests that these mutations impair the transfer of a proton from O-2 to O-1 at the initial enediolate phosphate intermediate. No products are detected for the LDM-catalyzed isomerization reactions in D2O of [1-13C]GA and HPi, but the L6RM-catalyzed reaction in the presence of 0.020 M dianion gives a 2% yield of the isomerization product [2-13C,2-2H]GA.

Reflections on the catalytic power of a TIM-barrel

“…69 These results provide a remarkable example of the plasticity of the structure of this TIM barrel, 74 where the severe distortion in structure of the unliganded wild type enzyme (Figure 5B) at the L6RM is overcome by the utilization of substrate binding energy to mold the enzyme into the active closed form.…”

“…40,73 No elimination reaction products are observed for the L6RM. 69 This shows that the L6RM-catalyzed reaction is through a catalytically active closed enzyme ( E C , Scheme 3B), which is stabilized towards elimination of PO 4 3− by interactions between loop 6 and the enediolate phosphate intermediate.…”

“…69 A comparison of the X-ray crystal structures for wild type c TIM (Figures 5A and 5B) and the L6RM (Figure 5B) shows that the mutation introduces a kink into loop 6 and a displacement of the side chain of Glu168 from the position of the tryptophan side chain of Trp168 for wild type TIM. This kink presumably reduces the destabilizing interactions between the carboxylate side chain of Glu168 and Glu129 (Figures 5A and 5B).…”

“…The transition state for the L6RM-catalyzed isomerization is stabilized by interactions with loop 6, since the activity of the L6RM is substantially higher than for a loop 6 deletion mutant (LDM). 40,69 The absence of a functioning loop at the LDM results in a change in the preferred catalyzed reaction from aldose-ketose isomerization to elimination of inorganic phosphate trianion to form methylgloxal. 40,73 No elimination reaction products are observed for the L6RM.…”

How an Inhibitor Bound to Subunit Interface Alters Triosephosphate Isomerase Dynamics

“…1 a) closes over the substrate and protects it from solvent exposure during catalysis. The specific closed state of loop 6 during formation of the substrate-enzyme complex is stabilized by the interaction between this so-called phosphate-gripper loop and the substrate phosphodianion (7). However, this loop closure is not ligand gated (8), i.e., its opening/closure has been observed in MD simulations on apo TIM (9) and its complex with DHAP (10).…”