Enzyme architecture: the effect of replacement and deletion mutations of loop 6 on catalysis by triosephosphate isomerase.
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.
Department of Chemistry, University at Buffalo , Buffalo, New York 14221, United States.
Xiang
X
Zhai
Maybelle K
MK
Go
AnnMarie C
AC
O'Donoghue
Tina L
TL
Amyes
Scott D
SD
Pegan
Yan
Y
Wang
J Patrick
JP
Loria
Andrew D
AD
Mesecar
John P
JP
Richard
D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D004099:Dihydroxyacetone Phosphate / Q000737:chemistry; D005986:Glyceraldehyde 3-Phosphate / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D005994:Glycerophosphates / Q000737:chemistry; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011817:Rabbits; D017384:Sequence Deletion
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D005994:Glycerophosphates; D005986:Glyceraldehyde 3-Phosphate; D004099:Dihydroxyacetone Phosphate; C029620:alpha-glycerophosphoric acid; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases