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Journal of Biochemistry Advance Access originally published online on January 23, 2008
Journal of Biochemistry 2008 143(5):625-632; doi:10.1093/jb/mvn007
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© 2008 The Japanese Biochemical Society.

Role of Disulphide Bonds in a Thermophilic Serine Protease Aqualysin I from Thermus aquaticus YT-1

Masayoshi Sakaguchi, Makoto Takezawa, Rie Nakazawa, Kazutaka Nozawa, Taro Kusakawa, Takeshi Nagasawa, Yasusato Sugahara and Masao Kawakita*

Department of Applied Chemistry, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo 192-0015, Japan

*To whom correspondence should be addressed. Tel: +81 42 628 4857, Fax: +81 42 628 5647, E-mail: bt13004{at}ns.kogakuin.ac.jp

Received October 30, 2007; Accepted January 17, 2008


  Abstract

A thermophilic serine protease, Aqualysin I, from Thermus aquaticus YT-1 has two disulphide bonds, which are also found in a psychrophilic serine protease from Vibrio sp. PA-44 and a proteinase K-like enzyme from Serratia sp. at corresponding positions. To understand the significance of these disulphide bonds in aqualysin I, we prepared mutants C99S, C194S and C99S/C194S (WSS), in which Cys69-Cys99, Cys163-Cys194 and both of these disulphide bonds, respectively, were disrupted by replacing Cys residues with Ser residues. All mutants were expressed stably in Escherichia coli. The C99S mutant was 68% as active as the wild-type enzyme at 40°C in terms of kcat value, while C194S and WSS were only 6 and 3%, respectively, as active, indicating that disulphide bond Cys163-Cys194 is critically important for maintaining proper catalytic site conformation. Mutants C194S and WSS were less thermostable than wild-type enzyme, with a half-life at 90°C of 10 min as compared to 45 min of the latter and with transition temperatures on differential scanning calorimetry of 86.7°C and 86.9°C, respectively. Mutant C99S was almost as stable as the wild-type aqualysin I. These results indicate that the disulphide bond Cys163-Cys194 is more important for catalytic activity and conformational stability of aqualysin I than Cys67-Cys99.

Key Words: disulphide bond, serine protease, subtilase, thermostability, activity

Abbreviations: DSC, differential scanning calorimetry; DTT, dithiothreitol; EDTA, ethylene diamine tetraacetic acid; IPTG, isopropyl β-D-thiogalactopyranoside; LB, Luria-Bertani; MES, 2-(N-morpholino)ethane sulphonic acid; N-sucAAPFpNA, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; ODA, oligonucleotide-directed dual amber; PMSF, phenylmethanesulphonyl fluoride; SPRK, proteinase K-like enzyme from Serratia sp.; TCA, trichloroacetic acid; VPR, proteinase from Vibrio PA-44


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