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

Zhai, X.; Go, M.K.; O'Donoghue, A.C.; Amyes, T.L.; Pegan, S.D.; Wang, Y.; Loria, J.P.; Mesecar, A.D.; Richard, J.P.

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.

Citation

Zhai, X., Go, M., O'Donoghue, A., Amyes, T., Pegan, S., Wang, Y., …Richard, J. (2014). Enzyme Architecture: The Effect of Replacement and Deletion Mutations of Loop 6 on Catalysis by Triosephosphate Isomerase. Biochemistry, 53(21), 3486-3501. https://doi.org/10.1021/bi500458t

• Journal Article Type: Article
• Acceptance Date: Apr 15, 2014
• Online Publication Date: May 22, 2014
• Publication Date: Jun 3, 2014
• Deposit Date: Oct 22, 2015
• Publicly Available Date: Feb 15, 2016
• Journal: Biochemistry.
• Print ISSN: 0006-2960
• Electronic ISSN: 1520-4995
• Publisher: American Chemical Society
• Peer Reviewed: Peer Reviewed
• Volume: 53
• Issue: 21
• Pages: 3486-3501
• DOI: https://doi.org/10.1021/bi500458t
• Public URL: https://durham-repository.worktribe.com/output/1397044