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

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• 作者：Xiang Zhai ; Maybelle K. Go ; AnnMarie C. O鈥橠onoghue ; Tina L. Amyes ; Scott D. Pegan ; Yan Wang ; J. Patrick Loria ; Andrew D. Mesecar ; John P. Richard
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：June 3, 2014
• 年：2014
• 卷：53
• 期：21
• 页码：3486-3501
• 全文大小：770K
• 年卷期：v.53,no.21(June 3, 2014)
• ISSN：1520-4995

文摘

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鈥?73 [Pompliano, D. L., et al. (1990) Biochemistry 29, 3186鈥?194] 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 k_cat/K_m 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鈥?3% 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 HP_i, 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.