Role of disulphide bonds in a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1.
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 degrees C in terms of k(cat) 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 degrees C of 10 min as compared to 45 min of the latter and with transition temperatures on differential scanning calorimetry of 86.7 degrees C and 86.9 degrees 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.
Sakaguchi
Masayoshi
M
Department of Applied Chemistry, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo 192-0015, Japan.
Takezawa
Makoto
M
Nakazawa
Rie
R
Nozawa
Kazutaka
K
Kusakawa
Taro
T
Nagasawa
Takeshi
T
Sugahara
Yasusato
Y
Kawakita
Masao
M
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002364:Caseins / Q000378:metabolism; D003553:Cystine / Q000737:chemistry*; D004795:Enzyme Stability; D009154:Mutation; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D013696:Temperature; D013824:Thermus / Q000201:enzymology*
D001426:Bacterial Proteins; D002364:Caseins; D003553:Cystine; D012697:Serine Endopeptidases; C055102:aqualysin I