Document Detail

Stopped-flow cryoenzymology: detection of catalytic intermediates not observable at ambient temperatures.
MedLine Citation:
PMID:  6584868     Owner:  NLM     Status:  MEDLINE    
Stopped-flow cryoenzymology has been used to study the reaction of porcine kidney cytosol leucine aminopeptidase [alpha-aminoacyl-peptide hydrolase(cytosol), EC] with L-leucylglycyldansyl hydrazide in 50% (vol/vol) methanol buffer over the -40 to 23 degrees C temperature range. Resonance energy transfer between tryptophan residues of the enzyme E and the dansyl group of the substrate S has been used to detect the formation and interconversion of reaction intermediates (E X S)i. Above 0 degrees C, a single intermediate E X S is formed and decays by first-order kinetics to products. However, at temperatures below -20 degrees C, a new intermediate (E X S)' is observed immediately after mixing, which relaxes to E X S within 100 msec. Because the detection of this new intermediate would not have been possible at ambient temperatures, this illustrates the value of stopped-flow cryoenzymology for studies of catalytic pathways.
H E Van Wart; S H Lin
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  80     ISSN:  0027-8424     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  1983 Dec 
Date Detail:
Created Date:  1984-05-11     Completed Date:  1984-05-11     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  7506-9     Citation Subset:  IM    
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MeSH Terms
Cytosol / enzymology
Kidney / enzymology
Leucyl Aminopeptidase / metabolism*
Grant Support
Reg. No./Substance:
EC Aminopeptidase

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