New Substrates for Enteropeptidase: I. Biologically Active Hepta-, Octa-, and Nonapeptides |
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Authors: | V. V. Likhareva B. V. Vas'kovskii N. E. Shepel' S. K. Garanin A. G. Mikhailova L. D. Rumsh |
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Affiliation: | (1) Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997, Russia |
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Abstract: | Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue if less than four negatively charged amino acid residues are in positions P2–P5 of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR VYIHPF) and the Hb(2–8) (LTAEEK A) and Hb(1–9) (MLTAEEK AA) peptides of the cattle hemoglobin -chain. The Km values for all the substrates ( 10–3 M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the –DDDDK– full-size linker (Km 10–4 M). The kcat values for AT and Hb(2–8) were also close and low ( 30 min–1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2–8) peptide by one amino acid residue from both its N- and C-termini results in a dramatic increase in the catalytic efficiency of the hydrolysis: the kcat value for Hb(1–9) is 1510 min–1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker. |
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Keywords: | chimeric proteins enteropeptidase peptide substrates trypsinogen |
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