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Journal of Biochemistry Advance Access originally published online on August 1, 2007
Journal of Biochemistry 2007 142(3):357-364; doi:10.1093/jb/mvm142
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© 2007 The Japanese Biochemical Society.

Roles of S3 Site Residues of Nattokinase on its Activity and Substrate Specificity

Shuming Wu1,2, Chi Feng2,3, Jin Zhong1,* and Liandong Huan3,{dagger}

1State Key Laboratory of Microbial Resource, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101; 2Graduate School of the Chinese Academy of Sciences, Beijing 100039; and 3Center for Metabolic Engineering of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R. China

*To whom correspondence should be addressed. Tel: +86-10-6480-7401, Fax: +86-10-6480-7401, E-mail: zhongj{at}im.ac.cn

{dagger}Correspondence may also be addressed. Tel: +86-10-6480-7439, Fax: +86-10-6480-7401, E-mail: huanld{at}sun.im.ac.cn

Received May 27, 2007; Accepted June 29, 2007


  Abstract

Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly100, Ser101 and Leu126) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly100 and Ser101 had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu126 might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3–S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.

Key Words: nattokinase, protease activity, S3 site, site-directed mutagenesis, substrate specificity

Abbreviations: NK, nattokinase, subtilisin NAT; suc-, succinyl; kcat, catalytic rate constant; t-PA, tissue-type plasminogen activator; DMF, N,N-dimethylformamide; ddH2O, double-distilled H2O; suc-AXPFpNA, succinyl-Ala-X-Pro-Phe-p-nitroanilide (where X represents the P3 amino acid); WT, wild-type nattokinase


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