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 (k(ass)) 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 k(ass) 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.
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.
Shun-ichi
S
Tanaka
Yuichi
Y
Koga
Kazufumi
K
Takano
Shigenori
S
Kanaya
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D017931:DNA Primers / Q000235:genetics; D019286:Endopeptidase K / Q000037:antagonists & inhibitors; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019766:Genes, Archaeal; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015843:Serpins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013381:Subtilisins / Q000037:antagonists & inhibitors; D019712:Thermococcus / Q000235:genetics / Q000378:metabolism*
D019843:Archaeal Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D013381:Subtilisins; D002918:Chymotrypsin; D019286:Endopeptidase K