4P61

CHICKEN TRIOSEPHOSPHATE ISOMERASE WITH LOOP6 MUTATIONS, V167P AND W168E.

Summary for 4P61

• Entry DOI: 10.2210/pdb4p61/pdb
• Related: 1TPH
• Descriptor: Triosephosphate isomerase (2 entities in total)
• Functional Keywords: triosephosphate isomerase, isomerase
• Biological source: Gallus gallus (Chicken)
• Total number of polymer chains: 2
• Total formula weight: 53196.70

Authors

Pegan, S.D.,Mesecar, A.D. (deposition date: 2014-03-21, release date: 2014-08-27, Last modification date: 2023-12-27)

Primary citation

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.
Enzyme architecture: the effect of replacement and deletion mutations of loop 6 on catalysis by triosephosphate isomerase.
Biochemistry, 53:3486-3501, 2014

PubMed 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-(13)C]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-(13)C,2-(2)H]GA.

PubMed: 24825099
DOI: 10.1021/bi500458t

Experimental method

X-RAY DIFFRACTION (1.34 Å)