Document Detail


Use of azidobestatin as a photoaffinity label to identify the active site peptide of leucine aminopeptidase.
MedLine Citation:
PMID:  1567860     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. They are found in most cells and tissues, and their activity has been implicated in myriad fundamental biochemical and physiological processes. Nevertheless, little is known about the structure of the aminopeptidase active sites. Beef lens leucine aminopeptidase (blLAP) can be considered prototypical of many enzymes in this family of peptidases. Bestatin, [(2S,3R)-(3-amino-2-hydroxy-4-phenyl-butanoyl)-L-leucine] is a nonhydrolyzable substrate analogue of a peptide, PheLeu, which is rapidly cleaved by blLAP. Bestatin incorporates elements of the putative tetrahedral intermediate, and this results in a greater than 10(5)-fold enhancement of binding relative to analogous peptides. Bestatin is the most tightly bound inhibitor of many aminopeptidases. Bestatin was successively converted to nitrobestatin, p-aminobestatin, [3H]-p-aminobestatin, and finally [3H]-p-azidobestatin (pAB). Like bestatin, pAB is a slow binding inhibitor of LAP (Ki*, the dissociation constant for the final complex, = approximately 4 x 10(-9); Ki, the dissociation constant for the initial collision complex, = approximately 10(-8). The t1/2 for binding of 2 x 10(-8) M and 8 x 10(-8) M bestatin are approximately 60 min and approximately 38 min, respectively. pAB, nitrobestatin, bestatin, and physiological peptides appear to bind in the same site, the first three with similar avidity. In the dark, pAB and bestatin protect low concentrations of the enzyme against inactivation upon extensive dialysis. The t1/2 for photoactivation of pAB is approximately 3 s. Irradiation of blLAP for such short periods of time resulted in insignificant change in activity. blLAP which was placed in 254-nm light in the presence of pAB was inactivated significantly. Treatment of photolabeled blLAP with trypsin produces only two peptides. Autoradiography and scintillation counting indicate that the active site is in the peptide which includes residues 138-487. Treatment of the same blLAP with hydroxylamine produces two different peptides, with the active site in the peptide 323-487. This indicates that the active site is in the carboxyl-terminal one-third of the protomer. It is likely that this photoaffinity label will be useful in identifying active sites in other aminopeptidases as well.
Authors:
A Taylor; C Z Peltier; E G Jahngen; E Laxman; Z Szewczuk; F J Torre
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  31     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1992 Apr 
Date Detail:
Created Date:  1992-05-28     Completed Date:  1992-05-28     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  4141-50     Citation Subset:  IM    
Affiliation:
Laboratory for Nutrition and Vision Research, USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111.
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MeSH Terms
Descriptor/Qualifier:
Affinity Labels*
Amino Acid Sequence
Azides / chemical synthesis,  metabolism*,  pharmacology
Binding Sites
Binding, Competitive
Hydroxylamine
Hydroxylamines / metabolism
Kinetics
Leucine / analogs & derivatives*,  chemical synthesis,  metabolism,  pharmacology
Leucyl Aminopeptidase / antagonists & inhibitors,  metabolism*
Molecular Sequence Data
Oligopeptides / metabolism
Photochemistry
Spectrophotometry
Trypsin / metabolism
Grant Support
ID/Acronym/Agency:
EY-08556/EY/NEI NIH HHS
Chemical
Reg. No./Substance:
0/Affinity Labels; 0/Azides; 0/Hydroxylamines; 0/Oligopeptides; 139915-04-1/azidobestatin; 58970-76-6/bestatin; 61-90-5/Leucine; 7803-49-8/Hydroxylamine; EC 3.4.11.1/Leucyl Aminopeptidase; EC 3.4.21.4/Trypsin

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


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