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

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Enteropeptidase (entero kinase) is a serine protease that cleaves proteins at the Asp-Asp-Asp-Asp-Lys sequence.
• The N-terminal amino acid of enteropeptidase light chain (EK(L)) is crucial for maintaining active site conformation.
• Understanding the role of the N-terminal residue can inform protein engineering strategies.

Purpose of the Study:

• To investigate the influence of N-terminal amino acid substitutions on enteropeptidase enzymatic activity.
• To explore the structural and functional implications of altering the N-terminus of EK(L).
• To provide a basis for engineering N-terminally modified enteropeptidase for expression in bacteria.

Main Methods:

• Construction and expression of 20 recombinant enteropeptidase light chain (rEK(L)) mutants in Saccharomyces cerevisiae.
• Enzymatic activity assays on the expressed rEK(L) mutants.
• Analysis of mutant kinetic properties and comparison with wild-type rEK(L).

Main Results:

• A single Ile --> Val substitution at the N-terminus of rEK(L) resulted in significant enzymatic activity.
• Kinetic properties of the Ile-to-Val mutant were comparable to the wild-type enzyme.
• The N-terminal Ile residue is oriented into a hydrophobic pocket, supporting the tolerance of Val substitution.

Conclusions:

• Valine can effectively substitute for isoleucine at the N-terminus of enteropeptidase without compromising enzymatic activity or active site conformation.
• The conserved N-terminal Ile or Val in trypsin-like serine proteases is functionally significant.
• This study provides a framework for N-terminal engineering of enteropeptidase for improved expression in Escherichia coli.