Mario Guarneri

Three-dimensional structure of the complex between pancreatic secretory trypsin inhibitor (Kazal type) and trypsinogen at 1.8 A resolution. Structure solution, crystallographic refinement and preliminary structural interpretation.

Abstract The three-dimensional structure of the proteic complex formed by bovine trypsinogen and the porcine pancreatic secretory trypsin inhibitor (Kazal type) has been solved by means of Patterson search techniques, using a predicted model of the trypsin-ovomucoid complex ( Papamokos et al., 1982 ). The structure of the complex, including 162 solvent molecules, has been refined at 1.8 A resolution (26,341 unique reflections) to a conventional crystallographic R factor of 0.195. The inhibitor molecule binds to trypsinogen via hydrogen bonds and/or apolar interactions at sites P9, P7, P6, P5, P3, P1, P1′, P2′ and P3′ of the contact area. The structure of the inhibitor itself resembles closely that of the third domain of Japanese quail ovomucoid inhibitor, recently reported by Weber et al. (1981) . The trypsinogen part of the complex resembles trypsin, as is the case in the trypsinogen-basic pancreatic trypsin inhibitor complex, but two segments of the activation domain adopt a different conformation. Most notably in the N-terminal region the Ile16-Gly19 loop, which is disordered in free trypsinogen and in the trypsinogen-basic pancreatic trypsin inhibitor complex ( Huber & Bode, 1978 ), assumes a regular structure and the polypeptide chain can be traced as far as residue Asp14. This new and fixed structure allows the formation of a buried salt link between the side-chains of Lys156 and Asp194. Conformations differing from those of trypsin are also found for residues 20 to 28 and residues 141 to 155. Some structural perturbation is observed in other parts of the molecule, including the calcium loop.

Interaction between serine (pro)enzymes, and kazal and kunitz inhibitors*

Equilibrium constants (Kd) for PSTI (porcine pancreatic secretory trypsin inhibitor: Kazal inhibitor) and BPTI (bovine basic pancreatic trypsin inhibitor: Kunitz inhibitor) binding to bovine β-trypsin, bovine trypsinogen, bovine α-chymotrypsin, human urinary kallikrein and porcine pancreatic kallikrein have been obtained at pH 8. Kd values have been determined by measuring the loss of the enzymatic activity and the spectral changes accompanying the formation of the PSTI: (pro)enzyme and BPTI: (pro)enzyme adducts. PSTI is characterized by systematically lower affinities than BPTI and does not inhibit either kallikrein up to inhibitor concentrations of 3 m m . As in the case of BPTI, the affinity of PSTI for bovine trypsinogen increases in the presence of 0·02 m -H-Ile-Val-OH, which mimics, in the proenzyme, the N-terminal segment of the activated bovine β-trypsin. BPTI: bovine trypsinogen: H-Ile-Val-OH and PSTI: bovine trypsinogen: H-Ile-Val-OH complexes show absorption spectra similar to those of BPTI: bovine β-trypsin and PSTI: bovine β-trypsin adducts. The pH dependence of the equilibrium constants for PSTI and BPTI binding to bovine β-trypsin, bovine trypsinogen and bovine α-chymotrypsin is roughly the same for both the inhibitors with the following scale of apparent affinities: β-trypsin ≫ α-chymotrypsin > trypsinogen. The simplest model that describes the experimental data, between pH 5 and 9·5, takes into account a single ionizable group with a pKUNL of approximately 7·2 in the free (pro)enzymes, which appears to decrease by about two units upon PSTI and BTPI binding. The most probable ionizing group to account for such a behaviour is His57, present at the active site of (pro)enzymes. Comparison of the three-dimensional structures of PSTI and BPTI adducts shows that the respective reactive sites, despite structural identity around the scissile peptide bond, differ in their geometry of subsites further away from it. In particular, the wider reactive site polypeptide loop of PSTI as compared to that of BPTI may be responsible, at least in part, for the different binding behaviour of the two inhibitors.

Inhibition of 'in vitro' tumor cell growth by aromatic polyamidines exhibiting antiproteinase activity.

Aromatic polyamidines containing two, three or four benzamidine residues inhibit proteinase activity and proliferation of different human tumor cell lines, including leukemic (K562, HEL), melanoma (Colo 38) and B-lymphoid (WI-L2) cell lines. In addition, the benzamidine derivatives analysed in the present study inhibit cell growth of the Chinese hamster FHO6T1-1 cell line, obtained after transfection of primary lung cells with the activated human T24-Ha-ras-1 oncogene.After treatment of FHO6T1-1 cells with benzamidine derivatives, a sharp decrease of the content of Ha-ras-1 mRNA was found, but not of transferrin receptor mRNA.We found that inhibition of cell proliferation by tetra-benzamidine derivatives is not restricted to tumor cells, but concerns also non-tumorigenic cell lines as well as normal primary fibroblasts. Therefore, our analysis was extended to di- and tri-benzamidine derivatives, which could be proposed as useful substrates in the synthesis of drug-conjugated monoclonal antibodies or growth factors. The data obtained demonstrate that these latter compounds and their halo-derivatives also exhibit strong antiproliferative effects onin vitro cultured cells.

Binding of the Recombinant Proteinase Inhibitor Eglin C from Leech Hzrudo Medzcznalzs to Human Leukocyte Elastase, Bovine α-Chymotrypsin and Subtilisin Carlsberg: Thermodynamic Study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human α-, β- and γ-thrombin; a kinetic and thermodynamic study

Abstract Kinetic and thermodynamic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human α-, β- and γ-thrombin have been determined, between 5 and 45°C, at pH 7.5. BPTI-binding properties to human thrombins have been analyzed in parallel with those of serine (pro)enzymes acting on cationic and non-cationic substrates, with particular reference to the bovine β-trypsin/BPTI system. The observed binding behaviour of BPTI to human α-, β- and γ-thrombin has been related to the inferred stereochemistry of the enzyme/inhibitor contact region(s).

Tetra-p-amidinophenoxy-propane as a probe of the specificity site of serine proteases

Enea MENEGATTI, Mario GUARNERI, Roberto FERRONI, Martin0 BOLOGNESI+, Paolo ASCENZI* and Eraldo ANTONINI*> Institute of Pharmaceutical Chemistry, Univ. of Ferrara, Via Scandiana 21,441OO Ferrara, +Institute of Crystallography, Univ. of Pavia, Vip Bassi 4,271OO Pavia, *CNR Center for Molecular Biology, Institutes of Chemistry and Biochemistry, Faculty of Medicine, Univ. of Rome, piazzale Aldo More 3, 00185 Rome, Italy

Catalytic and ligand binding properties of bovine trypsinogen and its complex with the effector dipeptide Ile-Val. A comparative study.

SummarySteady-state and pre-steady-state kinetic data for the trypsinogen catalyzed hydrolysis of a series of synthetic substrates (i.e. p-nitrophenyl esters of N-α-carbobenzoxy-L-amino acids) have been obtained as a function of pH (3.4–8). Moreover, the effect of ethylamine on the hydrolysis of a neutral substrate and benzamidine binding have been extensively studied.In order to obtain direct information on the transition of trypsinogen to a β-trypsin-like structure, the role of the effector dipeptide Ile-Val on the catalytic and ligand binding properties of the zymogen has been investigated. Kinetic and thermodynamic data for β-trypsin and α-chymotrypsin are also reported for the purpose of an homogeneous comparison of the various (pro)enzymes.Under all the experimental conditions, kinetic data for (pro)enzyme catalysis are consistent with the minimum three-step mechanism:

Synthesis of some isoxazole analogues of retinoids: biological effects toward tumor cell lines

E + S\mathop \rightleftharpoons \limits_{k_{ - 1} }^{k_{ + 1} } E.S\mathop \rightleftharpoons \limits_{k_{ - 2} }^{k_{ + 2} } \mathop E\limits_{\mathop + \limits_{P_1 } } .P\mathop \rightleftharpoons \limits_{k_{ - 3} }^{k_{ + 3} } E + P_{2,}