As for many enzymes, the enzymatic pathway of triosephosphate isomerase (TIM) includes the partially rate determining motion of an active site loop (loop 6, residues 166-176), which must remain closed during chemistry but must open just before product release. The motion of this loop was monitored using laser induced temperature-jump relaxation spectroscopy at nanosecond to millisecond time resolution. Trp168 in the hinge of the mobile loop served as a fluorophore reporter in a mutant of the yeast enzyme. The opening rate was studied as a function of the concentration of glycerol 3-phosphate, a substrate surrogate. Monoexponential kinetics were observed; assuming a simple two-step ligand release mechanism involving an encounter complex intermediate, the time scales of loop opening and closing were derived. The opening rate of the loop at 25 degrees C was determined to be 2500 +/- 1000 s(-1), in remarkable agreement with solution and solid state NMR measurements. The closing rate at the same temperature was 46,700 +/- 1800 s(-1). The rates were also studied as a function of the sample temperature following the jump. Enthalpies of activation of the loop motion, DeltaH(close) and DeltaH(open), were estimated to be 13.8 and 14.1 kcal/mol, respectively. The enthalpy of dissociation estimated from the kinetic studies is in reasonable agreement with steady-state values. Moreover, the enthalpy was dissected, for the first time, into components associated with ion binding and with protein conformational change. The enthalpy of the release reaction appeared to have a substantial contribution from the dissociation of the ligand from the encounter complex, found to be endothermic at 6 kcal/mol. In contrast, the population ratio of the open to closed loop conformations is found to favor the closed conformation but to be substantially less temperature dependent than the release step. Preliminary data of other ligands show that G3P behavior resembles that of the substrate but differs from 2-phosphoglycolate, a tight binding inhibitor, and phosphate. This study represents one of the first detailed comparisons between NMR and fluorescence based probes of protein motion and results in good agreement between the methods. The data in aggregate support a model in which the rate of the loop opening for TIM is dependent on the ligand and results in opening rates in the presence of the product that are comparable to enzymatic throughput, kcat.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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Evidence ID | Analyze ID | File | Description |
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