Document Detail

Inactivation of Staphylococcus hyicus lipase by hexadecylsulfonyl fluoride: evidence for an active site serine.
MedLine Citation:
PMID:  8029215     Owner:  NLM     Status:  MEDLINE    
The Staphylococcus hyicus lipase is an acyl hydrolase with broad substrate specificity including neutral glycerides and phospholipids. To obtain further insight into the mechanism of action of this enzyme, we tested several sulfonyl fluorides as active site-directed inhibitors. The enzyme is resistant to the well-known serine protease/esterase inhibitor phenylmethanesulfonyl fluoride (PMSF), but is rapidly inactivated by hexadecylsulfonyl fluoride. The kinetics of inactivation were studied in Triton X-100 micelles. Inactivation is fast and the rate of inactivation is constant over the pH range where this lipase is active. Metal ions like Ca2+ and Sr2+ do not appreciably influence the rate of inactivation, although the enzymatic activity is significantly increased, suggesting a structural role for these ions. The S. hyicus lipase contains a consensus sequence G-H/Y-S-X-G. Substitution by site-directed mutagenesis of this serine (Ser369) by a cysteine resulted in a mutant with only 0.2% residual activity. The activity of this mutant could not be inhibited with water-soluble sulfhydryl reagents either in the presence or absence of Triton X-100 micelles. In the presence of Triton X-100 micelles, inactivation of the mutant occurred with 4-nitrophenylhexadecyl disulfide (t1/2 = 125 min) while the wild-type enzyme does not react at all. We conclude that Ser369 is the active site residue and that in water this residue is inaccessible. Only after interfacial activation Ser369 (or Cys369) becomes exposed and reacts with irreversible inhibitors.
M Leuveling Tjeenk; Y B Bulsink; A J Slotboom; H M Verheij; G H de Haas; G Demleitner; F Götz
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Protein engineering     Volume:  7     ISSN:  0269-2139     ISO Abbreviation:  Protein Eng.     Publication Date:  1994 Apr 
Date Detail:
Created Date:  1994-08-08     Completed Date:  1994-08-08     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8801484     Medline TA:  Protein Eng     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  579-83     Citation Subset:  IM    
Department of Enzymology and Protein Engineering, University of Utrecht, The Netherlands.
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MeSH Terms
Bacterial Proteins / antagonists & inhibitors,  metabolism*
Binding Sites
Carboxylic Ester Hydrolases / antagonists & inhibitors,  metabolism*
Cations, Divalent / pharmacology
Disulfides / metabolism
Phenylmethylsulfonyl Fluoride / pharmacology
Recombinant Proteins / metabolism
Staphylococcus / enzymology*
Sulfones / pharmacology*
Reg. No./Substance:
0/4-nitrophenylhexadecyl disulfide; 0/Bacterial Proteins; 0/Cations, Divalent; 0/Disulfides; 0/Micelles; 0/Recombinant Proteins; 0/Sulfones; 329-98-6/Phenylmethylsulfonyl Fluoride; 56-45-1/Serine; 86855-26-7/hexadecanesulfonyl fluoride; 9002-93-1/Octoxynol; EC 3.1.1.-/Carboxylic Ester Hydrolases

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