Effect of calcium ions on enteropeptidase catalysis.

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 (Km 59.6 µM, kcat 6660 min−1-, kcat/Km 111 µM−1·min−1), as well as for enteropeptidase preparation with deleted 118–783 fragment of the heavy chain (Km 176.9 µM, kcat 6694 min−1, kcat/Km 37.84 µM−1·min−1) and trypsin (Km 56.0 µM, kcat 8280 min−1, kcat/Km 147.86 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.