Biochemical Characterization of Human Enteropeptidase Light Chain

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M. E. Gasparian^*, V. G. Ostapchenko, D. A. Dolgikh, and M. P. Kirpichnikov

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (7-495) 336-8011; E-mail: marine_gasparian@yahoo.com

^* To whom correspondence should be addressed.

Received July 7, 2005; Revision received October 17, 2005

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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 (K_m) 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/K_m 260 mM^-1·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.

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KEY WORDS: human enteropeptidase, recombinant fusion proteins, specificity, kinetic parameters, protease inhibitors

DOI: 10.1134/S0006297906020015

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