Marianne Pozsgay

Investigation of the substrate-binding site of human plasmin using tripeptidyl-p-nitroanilide substrates

Abstract The hydrolysis of tripeptidyl-p-nitroanilides by human plasmin /EC 3.4.21.7./ was studied and the kinetic parameters were determined. The individual contributions of the amino acid side chains at the P 1 -P 4 subsites to the kinetic parameters were calculated by regression analysis. The highest contributions yielded the structure of an “optimum” substrate, D-Ile-Phe-Lys-pNA. Its predicted kinetic parameters, K m = 9.6 × 10 -6 M and k cat /K m = 284 789 M -1 s -1 , appeared to be about 40 times as good as those of H-D-Val-Leu-Lys-pNA /S-2251/ applied for the determination of the plasminogen and plasmin content of blood in various laboratories. At the S 2 -S 4 segment in the binding site, which interacts with the P 2 -P 4 moieties of the substrate, plasmin favoured uniformly hydrophobic substituents.

Mapping of the substrate-binding site of the human granulocyte elastase by the aid of tripeptidyl-p-nitroanilide substrates

The kinetic properties of the human granulocyte elastase /EC 3.4.21.11/ were investigated with 24 tripeptidyl-pNA substrates. By the regression analysis of the kinetic data obtained with 15 substrates a relatively hydrophobic compound, Boc-D-Phe-Ala-Nle-pNA, was predicted as the optimal substrate sequence. The compound was synthesized, assayed and the predicted Km = 4.2 uM was confirmed experimentally. The substrate-binding site of granulocyte elastase appeared to be hydrophobic and very much similar to that of the pancreatic enzyme at the S2–S4 subsites, but the S1 subsite, which determines the primary specificity, could accomodate bulkier residues and it was less selective than that in the pancreatic enzyme.

Investigation on the substrate specificity of human plasmin using tripeptidyl-p-nitroanilide substrates.

The hydrolysis of 35 tripeptidyl-p-nitroanilides was studied with human plasmin and the kinetic parameters were determined. The individual contribution of the various side chains to the kinetic parameters was calculated by regression analysis. Considering Km, substrates having Z-D-Ile-Phe-Lys as well as H-D-Ile-Phe-Lys sequences were found to be the best, while Bz-Ile-Leu-Lys and pGlu-Leu-Lys sequences are the best for kcat. The Km values of substrates protected at N-terminus are lower, their kcat values are higher than those of the unprotected ones with the same sequence.

The interaction of heparin with human plasmin

1. The interaction of heparin with human plasmin was investigated measuring plasmin activity and enzyme inactivation in the presence of heparin. Hydrolysis of synthetic substrates (H-D-Val-Leu-Lys-pNA, H-D-Val-Phe-Lys-pNA and H-D-Pro-Phe-Lys-pNA) by plasmin was enhanced by heparin through an increase in kcat values. 2. This effect was the consequence of a change of Vmax since Km values were not altered in the presence of heparin. The polysaccharide also enhanced the rate of enzyme inactivation using TLCK as an active site blocking reagent. 3. Furthermore, heparin increased the heat sensitivity of plasmin, when synthetic substrate H-D-Val-Leu-Lys-pNA was used but it did not affect enzyme activity towards N-benzoyl-L-arginine-ethylester substrate. 4. The data show that microenvironmental conformation around the active center of plasmin is influenced by heparin.

Investigations on new tripeptidyl-p-nitroanilide substrates for subtilisins

The specificity of subtilisins (EC. 3.2.21.14) in splitting the peptide bonds at different amino acids of polypeptide chains is relatively broad, although X-ray data indicate that the substrate binding site favours non-polar amino acid side chains [ 1,2] . Several authors reported on the rate of hydrolysis of tripeptides composed of non-polar amino acids, e.g., Z-Gly-GlyLeu-NH2 [3] and its p-nitroanilide derivative [4]. In this paper investigations are described on nine tripeptidyl-p-nitroanilide substrates containing polar amino acid residues which increased both the solubility of compounds in aqueous media and the affinity of substrates compared to those studied by other authors [3,4] . The comparison of the Michaelis constants, Km, indicates that the compounds containing basic amino acid side chain at P1 binding site are just as good substrates as those with a non-polar side chain at the same site. Serva, Heidelberg, FRG. Kinetic investigations were carried out in 50 mM Tris-HCl buffer, pH 8.1. The relative proteolytic activities of subtilisins were measured on denatured hemoglobin [5] (table 1). Bz-Phe-Val-Arg-pNA (S-21 60) and H-D-ValLeu-Lys-pNA (S-2251) were obtained from AB Bofors, Nobel Division, Miilndal, Sweden. The other substrates (see table 1) were synthetized in our laboratory [6]. The substrates were dissolved in the buffer except Bz-Phe-Val-Arg-pNA which was dissolved in distilled water. Kinetic measurements were carried out at 37°C in a Unicam SP 500 spectrophotometer. The increase in absorption was recorded for 3-5 min at 405 nm where the absorption of substrates is negligible. The enzyme concentration varied between 0.725 nM and 4.35 PM. Initial velociti&s were calculated from the change in absorption using a molar absorption coefficient of 10 600 for p-nitroaniline.