Inhibition of chymotrypsin- and subtilisin-like serine proteases with Tk-serpin from hyperthermophilic archaeon Thermococcus kodakaraensis

A serpin homologue (Tk-serpin) from the hyperthermophilic archaeon Thermococcus kodakaraensis was overproduced in E. coli, purified, and characterized. Tk-serpin irreversibly inhibits Tk-subtilisin (TKS) from the same organism with the second-order association rate constants (kass) of 5.2 × 103 M− 1 s− 1 at 40 °C and 3.1 × 105 M− 1 s− 1 at 80 °C, indicating that Tk-serpin inhibits TKS more strongly at 80 °C than at 40 °C. It also irreversibly inhibits chymotrypsin, subtilisin Carlsberg, and proteinase K at 40 °C with the kass values comparable to that for TKS at 80 °C. Casein zymography showed that Tk-serpin inhibits these proteases by forming a SDS-resistant complex, which is typical to inhibitory serpins. The ratio of moles of Tk-serpin needed to inhibit 1 mol of protease (stoichiometry of inhibition, SI) varies from 40 to 80 at 20 °C, but decreases to the minimum values of 3–7 as the temperature increases. The inhibitory activities of Tk-serpin for these proteases increase as the stabilities of these proteases decrease, suggesting that a flexibility of the active-site of protease is one of the determinants for susceptibility of protease to inhibition by Tk-serpin. This report showed for the first time that Tk-serpin inhibits both chymotrypsin- and subtilisin-like serine proteases and its inhibitory activity increases as the temperature increases up to 100 °C.

Graphical AbstractTk‐serpin from the hyperthermophilic archaeon Thermococcus kodakaraensis was characterized. Tk‐serpin irreversibly inhibits Tk-subtilisin more strongly at 80°C than at 40°C. The inhibitory activity of Tk‐serpin increases as the temperature increases up to 100°C.Figure optionsDownload high-quality image (133 K)Download as PowerPoint slideResearch Highlights
► Tk-serpin from the hyperthermophilic archaeon Thermococcus kodakaraensis was characterized.
► Tk-serpin irreversibly inhibits Tk-subtilisin more strongly at 80 °C than at 40 °C.
► The inhibitory activity of Tk-serpin increases as the temperature increases up 100 °C.