[Mechanism of arginine deiminase activity by site-directed mutagenesis].

Abstract

Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

• Authors: Lifeng Li, Ye Ni, Zhihao Sun
• Journal: Sheng wu gong cheng xue bao = Chinese journal of biotechnology (Sheng Wu Gong Cheng Xue Bao) Vol. 28 Issue 4 Pg. 508-19 (Apr 2012) ISSN: 1000-3061 [Print] China
• PMID: 22803400 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Hydrolases
• arginine deiminase

Topics

• Escherichia coli (metabolism)
• Hydrolases (genetics, metabolism)
• Mutagenesis, Site-Directed