EFFECT OF TETHERED PEPTIDYLCHLOROMETHYLKETONE INHIBITORS ON THERMAL-STABILITY AND DOMAIN INTERACTIONS OF UROKINASE AND OTHER SERINE PROTEASES

The melting of several serine proteases that had been reacted with dif ferent peptidylchloromethylketone (cmk) inhibitors was studied by fluo rescence spectroscopy and calorimetry. These inhibitors, which cross-l ink the two domains of the proteases, invariably increased the melting temperature by as much as 28.5-degrees-C. The magnitude of the effect was dependent on the size and composition of the peptide moieties. Th e DELTAG of unfolding of tosyl-Phe-cmk-chymotrypsin was 13.5 kcal/mol compared to only 8.3 kcal/mol for chymotrypsin. Binding of cmk inhibit ors also protected the two interacting domains of urokinase from acid- induced decooperation and caused them to merge into a highly cooperati ve structure upon refolding at low pH. Fluorescence-detected melting c urves 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 uninhibite d enzyme. Upon second heating, the melting curves were similar to thos e 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, t he 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 t oward a second heating. At lower pH, second heating produced biphasic or triphasic melting curves that were attributed to differential proto nation of acid-titratable groups on the enzyme and/or inhibitor at the time of refolding. Similar effects were observed with other trypsin-l ike proteases, indicating that the hysteresis and bi- and triphasic re folding at low pH are rather general for this class of enzyme.