Biochemical characterization of human enteropeptidase light chain.
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.
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia. marine_gasparian@yahoo.com
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Gasparian
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Ostapchenko
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Dolgikh
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Kirpichnikov
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D002417:Cattle; D004765:Enteropeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D012964:Sodium; D004765:Enteropeptidase; D002118:Calcium