Key result
Trp-107 and Trp-253 mutations identify key residues driving stimulated pancreatic triglyceride lipase fluorescence.
Why the study?
Movement of the surface lid of human pancreatic triglyceride lipase is accompanied by a large change in steady state tryptophan fluorescence, but the underlying mechanism and specific tryptophan residues responsible had not been identified.
Identifies Trp-107 and Trp-253 as the specific residues responsible for the fluorescence change during lid opening of human pancreatic triglyceride lipase and demonstrates that PTL conformation in solution differs from its crystal structure.
Answers are grounded in this paper and cite their sources.
Does not alter clinical lipase use; extends PTL conformational insights beyond crystal structures.
The conformation of a surface loop, the lid, controls activity of pancreatic triglyceride lipase (PTL) by moving from a position that sterically hinders substrate access to the active site into a new conformation that opens and configures the active site. Movement of the lid is accompanied by a large change in steady state tryptophan fluorescence. Although a change in the microenvironment of Trp-253, a lid residue, could account for the increased fluorescence, the mechanism and tryptophan residues have not been identified. To identify the tryptophan residues responsible for the increased fluorescence and to gain insight into the mechanism of lid opening and the structure of PTL in aqueous solution, we examined the effects of mutating individual tryptophan residues to tyrosine, alanine, or phenylalanine on lipase activity and steady state fluorescence. Substitution of tryptophans 86, 107, 253, and 403 reduced activity against tributyrin with the largest effects caused by substituting Trp-86 and Trp-107. Trp-107 and Trp-253 fluorescence accounts for the increased fluorescence emissions of PTL that is stimulated by tetrahydrolipstatin and sodium taurodeoxycholate. The largest contribution is from Trp-107. Contrary to the prediction from the crystal structure of PTL, Trp-107 is likely exposed to solvent. Both tetrahydrolipstatin and sodium taurodeoxycholate are required to produce the increased fluorescence in PTL. Alone, neither is sufficient. Colipase does not significantly influence the conformational changes leading to increased emission fluorescence. Thus, Trp-107 and Trp-253 contribute to the change in steady state fluorescence that is triggered by mixed micelles of inhibitor and bile salt. Furthermore, the results suggest that the conformation of PTL in solution differs significantly from the conformation in crystals.
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Freie et al. (2009) studied this question. Site-directed mutagenesis of individual tryptophan residues (Trp-86, Trp-107, Trp-253, Trp-403) to tyrosine, alanine, or phenylalanine, stimulated by tetrahydrolipstatin and sodium taurodeoxycholate vs. Wild-type human pancreatic triglyceride lipase or single-agent treatment (tetrahydrolipstatin or sodium taurodeoxycholate alone) was evaluated on Steady state tryptophan fluorescence emission and lipase activity against tributyrin. Mutating Trp-107 and Trp-253 revealed they account for the increased fluorescence of pancreatic triglyceride lipase stimulated by tetrahydrolipstatin and sodium taurodeoxycholate.
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