Effect of tethered peptidylchloromethylketone inhibitors on thermal stability and domain interactions of urokinase and other serine proteases

Valery V. Novokhatny, Leonid V. Medved, Andrew Mazar, Kenneth C. Ingham*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

The melting of several serine proteases that had been reacted with different peptidylchloromethylketone (cmk) inhibitors was studied by fluorescence spectroscopy and calorimetry. These inhibitors, which crosslink the two domains of the proteases, invariably increased the melting temperature by as much as 28.5 °C. The magnitude of the effect was dependent on the size and composition of the peptide moieties. The ΔG of unfolding of tosyl-Phe-cmk-chymotrypsin was 13.5 kcal/mol compared to only 8.3 kcal/mol for chymotrypsin. Binding of cmk inhibitors also protected the two interacting domains of urokinase from acid-induced decooperation and caused them to merge into a highly cooperative structure upon refolding at low pH. Fluorescence-detected melting curves of Glu-Gly-Arg-cmk-urokinase indicated that unfolding/refolding at pH 4.5 is characterized by dramatic hysteresis; the cooling curves fell close to those obtained upon heating or cooling of the uninhibited enzyme. Upon second heating, the melting curves were similar to those of the original. The hysteresis effects are interpreted as follows. The tethered tripeptide binds to the active site, causing the protein to melt at much higher temperature in a single cooperative step, as if the two domains are merged into one cooperative unit. Upon cooling, the unfolded protein, with the inhibitor still attached, refolds at the same temperature as the underivatized protein. Only after the native structure is formed does the peptide moiety again bind and stabilize toward a second heating. At lower pH, second heating produced biphasic or triphasic melting curves that were attributed to differential protonation of acid-titratable groups on the enzyme and/or inhibitor at the time of refolding. Similar effects were observed with other trypsin-like proteases, indicating that the hysteresis and bi- and triphasic refolding at low pH are rather general for this class of enzyme.

Original languageEnglish (US)
Pages (from-to)17211-17218
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number23
StatePublished - 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Effect of tethered peptidylchloromethylketone inhibitors on thermal stability and domain interactions of urokinase and other serine proteases'. Together they form a unique fingerprint.

Cite this