The Role of Loop-Loop Interactions in Coordinating Motions and Enzymatic Function in Triosephosphate Isomerase

摘要

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 homolog caused a 10²-fold loss in enzymatic activity, a decrease in substrate binding affinity and a decrease in thermal stability. Isotope exchange studies performed by one-dimensional ¹H 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 R1ρ experiments indicate that upon mutation of loop 7, the chemical exchange rate for active site loop motion is nearly doubled and the coordinated motion of loop 6 is reduced relative to 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.