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-α-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 (Km59.6 μM, kcat6660 min-1-, kcat/Km111 μM-1•min-1), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (Km176.9 μM, kcat6694 min-1, kcat/Km37.84 μM-1•min-1) and trypsin (Km56.0 μM, kcat8280 min-1, kcat/Km147.86 μ M-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 β-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions. © 2005 Pleiades Publishing, Inc.