Role of loop-loop interactions in coordinating motions and enzymatic function in triosephosphate isomerase.
The enzyme triosephosphate isomerase (TIM) has been used as a model system for understanding the relationship between protein sequence, structure, and biological function. The sequence of the active site loop (loop 6) in TIM is directly correlated with a conserved motif in loop 7. Replacement of loop 7 of chicken TIM with the corresponding loop 7 sequence from an archaeal homologue caused a 10(2)-fold loss in enzymatic activity, a decrease in substrate binding affinity, and a decrease in thermal stability. Isotope exchange studies performed by one-dimensional (1)H NMR showed that the substrate-derived proton in the enzyme is more susceptible to solvent exchange for DHAP formation in the loop 7 mutant than for WT TIM. TROSY-Hahn Echo and TROSY-selected R(1rho) experiments indicate that upon mutation of loop 7, the chemical exchange rate for active site loop motion is nearly doubled and that the coordinated motion of loop 6 is reduced relative to that of the WT. Temperature dependent NMR experiments show differing activation energies for the N- and C-terminal hinges in this mutant enzyme. Together, these data suggest that interactions between loop 6 and loop 7 are necessary to provide the proper chemical context for the enzymatic reaction to occur and that the interactions play a significant role in modulating the chemical dynamics near the active site.
Wang
Yan
Y
Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA.
Berlow
Rebecca B
RB
Loria
J Patrick
JP
D000818:Animals; D002645:Chickens; D004795:Enzyme Stability; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy; D009068:Movement; D009154:Mutation; D013696:Temperature; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D007554:Isotopes; D014305:Triose-Phosphate Isomerase