Document Detail

Time-resolved fluorescence allows selective monitoring of Trp30 environmental changes in the seven-Trp-containing human pancreatic lipase.
MedLine Citation:
PMID:  14580194     Owner:  NLM     Status:  MEDLINE    
Human pancreatic lipase (HPL, triacylglycerol acylhydrolase, EC is a carboxyl esterase which hydrolyzes insoluble emulsified triglycerides and is essential for the efficient digestion of dietary fats. Though the three-dimensional structure of this enzyme has been determined, monitoring the conformational changes that may accompany the binding of various substrates and inhibitors is still of interest. Because of its sensitivity and ease of use, fluorescence spectroscopy of the intrinsic Trp residues is ideally suited for this purpose. However, the presence of seven Trp residues spread all over the HPL structure renders the interpretation of the fluorescence changes difficult with respect to the identification and location of the conformational or environmental changes taking place at the various Trp residues. In this context, the aim of this work was to investigate the contribution of the individual Trp residues to the fluorescence properties of HPL. To this end, we analyzed the steady-state and time-resolved fluorescence parameters of five single-point mutants in which one Trp residue was substituted with a weakly fluorescent Phe residue. In addition to the Trp residues at positions 30, 86, and 252, strategically located with respect to the active site, we also mutated Trp residues at positions 17 and 402, as representative residues of the HPL N- and C-terminal domains, respectively. Taken together, our data suggested that the solvent-exposed Trp30 residue contributed to at least 44% of the overall fluorescence of wild-type HPL. Moreover, we found that the long-lived fluorescence lifetime (6.77 ns) of wild-type HPL could be specifically attributed to Trp30, a feature that enables selective monitoring of its environmental changes. Additionally, Trp residues at positions 17 and 402 strongly contributed to the 1.61 ns lifetime of HPL, while Trp residues at positions 86 and 252 contributed to the 0.29 ns lifetime.
Paul Ramos; Thierry Coste; Etienne Piémont; Jean Marc Lessinger; Jean Alain Bousquet; Catherine Chapus; Brigitte Kerfelec; Georges Férard; Yves Mély
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemistry     Volume:  42     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2003 Nov 
Date Detail:
Created Date:  2003-10-28     Completed Date:  2003-12-10     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  12488-96     Citation Subset:  IM    
Laboratoire de Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR 7034 CNRS, Faculté de Pharmacie, Université Louis Pasteur, Illkirch, France.
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MeSH Terms
Base Sequence
Cloning, Molecular
DNA Primers
Lipase / chemistry*,  genetics
Mutagenesis, Site-Directed
Pancreas / enzymology*
Point Mutation
Spectrometry, Fluorescence
Tryptophan / chemistry*
Reg. No./Substance:
0/DNA Primers; 73-22-3/Tryptophan; EC

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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