Investigation of the Substrate-Binding Site of Trypsin by the Aid of Tripeptidyl-<em>p</em>-nitroanilide Substrates.

The kinetic parameters of the tryptic hydrolysis of tripeptidyl-p-nitroanilide substrates were determined and the data were studied by regression analysis. The sequence of substrates optimal from the viewpoint of kinetic constants 1/K_m, k_cat and k_cat/K_m was established and the influence of amino acid side chains on the binding and reactivity of substrates was calculated. At subsite P₃ At subsite P₃[notation of Schechter and Berger (1967) Biochem, Biophys, Res. Commun. 27, 157] polar side chains (Asn, D-Arg) are favourable as regards 1/K_m, whereas hydrophobic side chains are preferred definitely from the viewpoint of catalytic efficiency, just as at subsite P₂. In the side chain contributions, calculated for the kinetic parameters, the P₃-S₃ interaction predominates, in spite of the fact that the properties of the residue at subsite P_i decide whether hydrolysis occurs at all. The ZAsn-Ile-Arg-Nan sequence was predicted as a better substrate than those tested experimentally. The compound was synthesized, and the calculated value of its 1/K_m (116.4 mM^-1) was in a good agreement with the measured value (100.2 mM^-1). Comparing the data obtained with trypsin with those observed with thrombin, elastase and substrates, we can establish that the homology of these enzymes can be characterized at each binding subsite by the aid of tripeptidyl- p-nitroanilide substrates. The quantities derived allow one to envisage a novel type of comparison of the proteases. [ABSTRACT FROM AUTHOR]