Effect of Calcium Ions on Enteropeptidase Catalysis

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A. G. Mikhailova^*, V. V. Likhareva, I. A. Prudchenko, and L. D. Rumsh

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (7-095) 335-7103; E-mail: anna@enzyme.siobc.ras.ru

^* To whom correspondence should be addressed.

Received December 2, 2004

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The effects of calcium ions on hydrolysis of low molecular weight substrates catalyzed by different forms of enteropeptidase were studied. A method for determining activity of truncated enteropeptidase preparations lacking a secondary trypsinogen binding site and displaying low activity towards trypsinogen was developed using N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25°C were determined for natural enteropeptidase (K_m 59.6 µM, k_cat 6660 min^-1, k_cat/K_m 111 µM^-1*min^-1), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (K_m 176.9 µM, k_cat 6694 min^-1, k_cat/K_m 37.84 µM^-1*min^-1) and trypsin (K_m 56.0 µM, k_cat 8280 min^-1, k_cat/K_m 147.86 µM^-1*min^-1). It was shown that the enzymes with trypsin-like primary active site display similar hydrolysis efficiency towards Z-Lys-S-Bzl. Calcium ions cause 3-fold activation of hydrolysis of the substrates of general type GD₄K-X by the natural full-length enteropeptidase. In contrast, the hydrolysis of substrates with one or two Asp/Glu residues at P2-P3 positions is slightly inhibited by Ca²⁺. In the case of enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 fragment), the activating effect of calcium ions was not detected for all the studied substrates. The results of hydrolysis experiments with synthetic enteropeptidase substrates GD₄K-F(NO₂)G, G₅DK-F(NO₂)G (where F(NO₂) is p-nitrophenyl-L-phenylalanine residue), and GD₄K-Nfa (where Nfa is beta-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.

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KEY WORDS: enteropeptidase, calcium ion, trypsinogen, activation, autolysis, trypsin, peptide substrates, fusion proteins

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