Enteropeptidase (enterokinase) is a serine protease highly specific for recognition and cleavage of the target sequence of Asp-Asp-Asp-Asp-Lys (D4K). The three-dimensional structure of the enteropeptidase shows that the N-terminal amino acid is buried inside the protein providing molecular interactions necessary to maintain the conformation of the active site. To determine the influence of the N-terminal amino acid of enteropeptidase light chain (EKL) on the enzymatic activity, we constructed various mutants including 17 different single amino acid substitutions and three different extensions at the N-terminal end. The mutants of recombinant enteropeptidase (rEKL) were expressed in Saccharomyces cerevisiae and secreted into culture medium. Among 20 different mutants tested, the only mutant with the Ile → Val substitution exhibited significant activity. The kinetic properties of the mutant protein were very similar to those of the wild-type rEKL. Based on the three-dimensional structure where the N-terminal Ile is oriented into hydrophobic pocket, the results suggest that Val could substitute Ile without affecting the active conformation of the enzyme. The results also explain why all trypsin-like serine proteases carry either Ile or Val at the N-termini and none other amino acid residues are found. Moreover, this finding provides a mental framework for expressing the N-terminally engineered enteropeptidase in Escherichia coli, utilizing the known property of the methionine aminopeptidase that exhibits poor activity toward the N-terminal Met-Ile bond, but offers efficient cleavage of the Met-Val bond.
Bibliographical noteFunding Information:
This work was supported, in part, by the Korea Science and Engineering Foundation (KOSEF) through the Bioproducts Research Center at Yonsei University (Grant No. 2000-2-0177) and by the Ministry of Commerce, Industry and Energy (IMT 2000 Grant and Clean Technology Grant). Authors express sincere thanks to Dr. J. H. Youn in IDRTech for critical reading of the paper.
All Science Journal Classification (ASJC) codes
- Molecular Biology