The kinetic behaviour of human thrombin has been studied with 26 tripeptidyl-p-nitroanilide substrates protected at the N terminus and with 9 unprotected ones. By the regression analysis of experimentally determined I/&,, k,,, and /cCat/Km values the individual contribution of each side chain of the various substrates to the kinetic parameters was calculated.The contributions to the kinetic parameters of the best substrates provide information about the structure of the binding site. The interaction of subsites S1 and PI, which determines primary specificity, proved to be marginal on the basis of contribution values, though it depends upon this contact whether the substrate is hydrolyzed at all. At subsite Sz proline appeared to be favourable. Subsite S3 plays an important role in efficiency. The best parameters were obtained here with the D configurations of bulky amino acid residues. The aromatic protecting groups applied did not improve the properties of substrates. BzDPhe-Pro-Arg-Nan was predicted by calculation to be better than the protected substrates assayed. The compound was synthesized and tested. Its experimentally determined l/K,,,, 55.1 mM-', was in good agreement with 50.9 mM-' found by calculation.Several serine proteases, e.g. thrombin, trypsin, plasmin and factor Xa, hydrolyze peptide bonds at the carboxylic group of basic amino acids. Their sensitivities to substrate specificity are, however, different. Among them thrombin is the most specific which, apart from a few exceptions, splits next to arginine [1,2] [9] and growth hormones [lo]. According to Muszbek [ I l l in actin it also hydrolyzes next to lysine, whereas hydrolysis does not occur next to ornithine [12].The detailed structure of the substrate-binding sites of thrombin is not yet known. The investigations on the specificity and substrate-binding sites have so far been carried out, in addition to fibrinogen, with peptide derivatives analogous to fibrinogen sequences [13,14] [2,16] and similar hydrophobic side chains, whereas results concerning the S3 substrate-binding subsite are contradictory. Scheraga holds that S3 is rather narrow; it is conceivable, though, that more than one binding domain is operative against long peptides [12].In an attempt to study the substrate-binding sites of thrombin we applied a mathematical method [19], by the aid of which, through the regression analysis of the kinetic constants measured for various tripeptidyl-p-nitroanilide substrates, the interaction between substrate side chains and the

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 l/Km, k,,, and k,,,/K, was established and the influence of amino acid side chains on the binding and reactivity of substrates was calculated.At subsite P3 [notation of Schechter and Berger (1967) Biochern. Biophys. Res. Commun. 27, 1571 polar side chains (Asn, D-Arg) are favourable as regards l/Km, whereas hydrophobic side chains are preferred definitely from the viewpoint of catalytic efficiency, just as at subsite Pz. In the side chain contributions, calculated for the kinetic parameters, the P3 -SJ interaction predominates, in spite of the fact that the properties of the residue at subsite PI 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 l/Km (116.4 mM-') was in a good agreement with the measured value (100.2 mM-').Comparing the data obtained with trypsin with those observed with thrombin, elastase and subtilisin, we can establish that the homology of these enzymes can be characterized at each binding subsite by the aid of tripeptidylp-nitroanilide substrates. The quantities derived allow one to envisage a novel type of comparison of the proteases.Among proteolytic enzymes favouring basic amino acid residues, and therefore cleaving preferentially at the carboxyl function of arginyl and lysyl side chains, trypsin has been studied in greatest detail. The chemistry of active sites [I] and the overall folding of polypeptide chains [2] are very similar in the various serine proteases, nevertheless these enzymes differ from one another in respect of substrate specificity.The structure of the substrate-binding site of trypsin has so far been studied by a variety of methods. It has been shown by X-ray crystallography how the enzyme binds polypeptide inhibitors of natural origin [3]. parison of these results with those obtained with other serine proteases renders it possible to draw conclusions not only about the substrate-binding site of trypsin but also concerning the primary specificity of serine proteases and the general features of substrate-binding subsites. MATERIALS A N D METHODS Assay of Enzyme ActivityCrystallized, lyophilized bovine pancreas trypsin was purchased from Merck (Darmstadt, FRG, lot no. 7901268). The enzyme was found to be homogenous by polyacrylamide gel electrophoresis. Trypsin solutions were freshly prepared in 1.0 mM hydrochloric acid. The amount of active enzyme was 87 %, as determined by active-site titration according to Chase and Shaw [12].Enzyme activity was measured in 50 mM Tris/HCl buffer, pH 8.1, at 37°C for 3 min. Enzyme and substrate concentrations were adjusted so that within this time interval the absorbance recorded at 405nm changed linearly in time. It was checked by thin-layer adsorption chr...

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