Biochemical characterization of human enteropeptidase light chain

Abstract

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.

A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD4K-conjugated 7-amino-4-methylcoumarin

“…Next, we determined the kinetic parameters of enteropeptidase for GD4K-AMC. As a control experiment, GD4K-NA was used as a substrate for bovine enteropeptidase light chain and the results agreed with those of previous studies (9)(10)(11), with the KM of GD4K-NA being 0.5 to 0.6 mM. The initial reaction rates of GD4K-AMC hydrolysis were measured and plotted on the Lineweaver-Burk plot (Fig.…”

Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli

“…Enterokinase recognizes Asp-Asp-Asp-Asp-Lys sequence and cleaves the C-terminal peptide bond of the lysine residue. Due to broad reaction scope (temperature range: 4-45°C; pH range: 4.5-9.5) and mild reaction conditions, enterokinase is considered a powerful tool and widely applied in biochemistry and biotechnology (Gasparian et al 2006). The source of natural enterokinase is limited due to high cost of extraction and separation, and the enzyme derived from the animals is always contaminated by other protease, which lead to degrade the protein (Vozza et al 1996).…”

“…There are reports on the use of prokaryotic or eukaryotic expression system to produce recombinant bovine (Kitamoto et al 1994;Collins-Racie et al 1995;Huang et al 2007) and mice (Sun et al 2006) enterokinase light chain protein with biological activity. However, there is lack of study on the recombinant human enterokinase light chain (Gasparian et al 2006). The human enterokinase light hEK L gene has been expressed in yeast cells (Pepeliaev et al 2011).…”

Human enteropeptidase light chain: Bioengineering of recombinants and kinetic investigations of structure and function

“…CaCl 2 enhances EP L activity at the 0.02 mM concentration. 26 NaCl was excluded because it has been shown to reduce EP L activity for GD 4 K-na. 26 As a result of the lim- (Table I); furthermore, the estimate reported here for Tag*off TM with GD4K-pNA is similar to the k cat reported elsewhere for hEP L with the substrate GD 4 K-na.…”

“…26 NaCl was excluded because it has been shown to reduce EP L activity for GD 4 K-na. 26 As a result of the lim- (Table I); furthermore, the estimate reported here for Tag*off TM with GD4K-pNA is similar to the k cat reported elsewhere for hEP L with the substrate GD 4 K-na. 26 For the single residue substrate Z-Lys-SBzl, no statistically significant differences were observed among the K M values; likewise, the k cat value for R96Q is only 1.1 times that for Y174R (P < 0.05) (Table I).…”