Engineered recombinant enteropeptidase catalytic subunit: Effect of N-terminal modification

Hye Won Song, Sung Il Choi, Baik Lin Seong

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1-6
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume400
Issue number1
DOIs
Publication statusPublished - 2002 Jan 1

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Enteropeptidase
Catalytic Domain
Amino Acids
trypsinogen activation peptide
Conformations
Substitution reactions
Aminopeptidases
Molecular interactions
Serine Proteases
Mutant Proteins
Amino Acid Substitution
Methionine
Yeast
Escherichia coli
Saccharomyces cerevisiae
Culture Media
Light
Kinetics
Enzymes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

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abstract = "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.",
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Engineered recombinant enteropeptidase catalytic subunit : Effect of N-terminal modification. / Song, Hye Won; Choi, Sung Il; Seong, Baik Lin.

In: Archives of Biochemistry and Biophysics, Vol. 400, No. 1, 01.01.2002, p. 1-6.

Research output: Contribution to journalArticle

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