Journal
BIOCHEMISTRY
Volume 48, Issue 21, Pages 4548-4556Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi9002887
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Funding
- NIH [R01-GM070823, 5T32GM008283]
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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 H-1 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-1 rho 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.
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