Mechanism of slow-binding inhibition of human leukocyte elastase by trifluoromethyl ketones

Mechanism of slow-binding inhibition of human leukocyte elastase by trifluoromethyl ketones

Kinetics of inhibition have been determined for the interaction of human leukocyte elastase (HLE) with two series of peptide trifluoromethyl ketones (TFMKs): X-Val-CF3,X-Pro-Val-CF3,X-Val-Pro-Val-CF3, and X-Lys(Z)-Val-Pro-Val-CF3, where X is MeOSuc or Z. These compounds are "slow-binding"...

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Bibliographic Details
Published in:Biochemistry (Easton) Vol. 26; no. 10; pp. 2682 - 2689
Main Authors: Stein, Ross L, Strimpler, Anne M, Edwards, Phillip D, Lewis, Joseph J, Mauger, Russel C, Schwartz, Jack A, Stein, Mark M, Trainor, D. Amy, Wildonger, Richard A, Zottola, Mark A
Format: Journal Article
Language:English
Published: Washington, DC American Chemical Society 19-05-1987
Subjects:
Analytical, structural and metabolic biochemistry
Biological and medical sciences
elastase
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Humans
Hydrogen-Ion Concentration
Hydrolases
Ketones - pharmacology
Kinetics
leukocytes
Leukocytes - enzymology
Mathematics
Oligopeptides - pharmacology
Pancreatic Elastase - antagonists & inhibitors
Pancreatic Elastase - blood
Structure-Activity Relationship
Thermodynamics
trifluoromethyl ketone
Human
Elastase
Inhibition
Enzyme
Leukocyte
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Summary:Kinetics of inhibition have been determined for the interaction of human leukocyte elastase (HLE) with two series of peptide trifluoromethyl ketones (TFMKs): X-Val-CF3,X-Pro-Val-CF3,X-Val-Pro-Val-CF3, and X-Lys(Z)-Val-Pro-Val-CF3, where X is MeOSuc or Z. These compounds are "slow-binding" inhibitors of HLE and, thus, allow the determination of Ki, the dissociation constant for the stable complex of inhibitor and enzyme, as well as kon and koff, the rate constants for formation and decomposition of this complex. Maximal potency is reached with Z-Lys(Z)-Val-Pro-Val-CF3, which displays a Ki less than 0.1 nM. Upon binding to HLE, these compounds undergo addition by the hydroxyl of the active site serine to form a hemiketal. The evidence supporting a hemiketal intermediate includes Ki values of 1.6 and 80,000 nM for Z-Val-Pro-Val-CF3 and its alcohol analogue, linear free energy correlations between inhibitory potency and catalytic efficiency for structurally related TFMKs and substrates, and the pH dependence of kon for the inhibition of HLE by Z-Val-Pro-Val-CF3, which is sigmoidal and displays a pKa of 6.9. Hemiketal formation is probably not rate limiting, however. Kinetic solvent isotope effects of unity suggest that kon cannot be rate limited by a reaction step, like hemiketal formation, that is subject to protolytic catalysis. A general mechanism that is consistent with these results is one in which formation of the hemiketal is rapid and is followed or preceded by a slow step that rate limits kon.
Bibliography:istex:8928FE26CFF6AD674F7C5D66137C33544492195C
ark:/67375/TPS-M1369CGZ-2
ObjectType-Article-1
SourceType-Scholarly Journals-1
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ISSN:0006-2960
1520-4995
DOI:10.1021/bi00384a005