Engineered recombinant enteropeptidase catalytic subunit: effect of N-terminal modification.
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 (EK(L)) 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 (rEK(L)) 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 rEK(L). 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.
Song
Hye-Won
HW
Protheon Incorporated, Yonsei Engineering Center B120E, Seoul 120-749, Korea.
Choi
Sung-Il
SI
Seong
Baik L
BL
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D020134:Catalytic Domain; D002417:Cattle; D002845:Chromatography; D004151:Dipeptides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000737:chemistry*; D005822:Genetic Vectors; D006801:Humans; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors; D014633:Valine / Q000737:chemistry
D004151:Dipeptides; D011994:Recombinant Proteins; D007532:Isoleucine; D004765:Enteropeptidase; D014633:Valine