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Biomedical subjects

E Menegatti

Publications and source records attributed to E Menegatti.

At least 109 records · Page 6Linked to original sources
PubMed1985

Ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids: synthesis and antiproteolytic activity towards bovine trypsin and porcine pancreatic kallikrein.

Ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids (namely, glycine, alanine, valine, leucine and phenylalanine) were synthesized and their inhibitory effect on the bovine trypsin and porcine pancreatic kallikrein catalyzed hydrolysis of p-nitroanilides of amino acids was investigated, at pH 8.1 and 37 degrees, in parallel with the effect of ethyl and/or methyl esters of N-benzoyl(-L-)amino acids and benzamidine. For both proteinases, the inhibitory effect of ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids is independent of the aminoacidic side chain, is closely similar to that of benzamidine (which binds at the S1 subsite of the proteinases examined and is commonly taken as a molecular inhibitor-model), and is higher by at least 10-fold than that of ethyl and/or methyl esters of N-benzoyl(-L-)amino acids (depending on the aminoacidic residue). On structural grounds, the peculiar inhibitory behaviour of ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids has been related to the interaction of the positively charged substituent at the N-position with the Asp189 residue present in the primary specificity subsite (S1) of bovine trypsin and porcine pancreatic kallikrein. The consistently lower affinity for porcine pancreatic kallikrein of all the inhibitors considered, as compared to bovine trypsin, may be related to the marked structural differences of the primary specificity subsite of these two serine proteinases.

Amidines↗
PubMed1984

Different primary specificity of porcine pancreatic beta-kallikrein-B and bovine beta-trypsin. A comparative steady-state and pre-steady-state study.

The values of pre-steady-state and steady-state parameters for the beta-trypsin catalyzed hydrolysis of Z-Arg-ONp and Z-Lys-ONp are superimposable between pH 2.4 and 8. At variance, the kinetic parameters for the beta-kallikrein-B catalyzed hydrolysis of Z-Arg-ONp are more favourable than those observed for Z-Lys-ONp and depend on different pKa values. The different primary specificity and the catalytic behaviour of beta-trypsin and beta-kallikrein-B reflect structural differences at their S1 subsite, especially at level of the 226 residue as well as the 217-220 segment.

Animals↗
PubMed1984

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human urinary kallikrein and to porcine pancreatic beta-kallikreins A and B.

The effect of pH and temperature on the association equilibrium constant (Ka) for bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B has been investigated. Ka values decrease with decreasing pH, reflecting the acid-midpoint and pK shifts, upon BPTI binding, of a three-proton co-operative transition, between pH 3 and 5, and of a single ionizable group, between pH 5 and 9. At pH 8, the values of delta H degree (between 7 degrees C and 42 degrees C) and delta S degree (at 21 degrees C) for BPTI binding to the glandular kallikreins considered were determined. In particular, the delta H degree values have been found to be independent of temperature and the following values have been obtained by van't Hoff plots: +1.8 kcal/mol, +2.3 kcal/mol and +2.4 kcal/mol (1 kcal = 4184 J) for the inhibitor binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B, respectively. Considering the known molecular structures of free porcine pancreatic beta-kallikrein A and BPTI, and of their complex, the stereochemistry of the enzyme : inhibitor contact regions was analysed for the three serine proteinases, in relation to their respective types of behaviour.

Animals↗
PubMed1984

The pH dependence of pre-steady-state and steady-state kinetics for the porcine pancreatic beta-kallikrein-B-catalyzed hydrolysis of N-alpha-carbobenzoxy-L-arginine p-nitrophenyl ester.

Pre-steady-state and steady-state kinetics of the porcine pancreatic beta-kallikrein-B (EC 3.4.21.35) catalyzed hydrolysis of ZArgONp have been determined between pH 2.4 and 8. The results are consistent with a minimum three-step mechanism involving an acyl-enzyme intermediate: (see formula). The formation of the E X S complex may be regarded as a pseudoequilibrium process; the minimum values for k+1 are 5.9 X 10(6) M-1 X s-1 (pH 5.5) and 9.4 X 10(5) M-1 X s-1 (pH 2.4) and that for k-1 is 600 s-1. The value of k-1/k+1 (= Ks) changes from 102 microM at pH greater than or equal to 5.5 to 638 microM at pH less than 2.4. The pH dependence of k+2 conforms to two ionizing groups, in the E X S complex, with pKa values of 3.4 +/- 0.1 and 7.05 +/- 0.10. The pH profile of k+2/Ks (= kcat/Km) reflects the ionization of two groups, in the free enzyme, with pKa values of 4.2 +/- 0.1 and 7.05 +/- 0.10. The pH dependence of k+3 implicates two ionizing groups in the deacylation step with pKa values of 4.6 +/- 0.1 and 7.0 +/- 0.1. At acid pH values (pH 2.4-4.4), k+3 is rate-limiting in catalysis, whereas for pH values higher than 4.4, k+2 becomes rate-limiting. The observed neutral and acid ionizations probably reflect the acid-base equilibrium of His-57 and Asp-189 involved in the central site of beta-kallikrein-B. The structural basis for the specificity and catalytic behaviour of this proteinase are discussed and a role for Ser-226 is pinpointed.

Animals↗
PubMed1984

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

Steady-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-alpha-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 beta-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 beta-trypsin and alpha-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: (formula; see text) involving the acyl intermediate E X P. In the presence of Ile-Val dipeptide, trypsinogen assumes catalytic and ligand binding properties that are reminiscent of activated beta-trypsin. This is at variance with free trypsinogen, which shows a alpha-chymotrypsin-like behavior. The large differences in the results of kinetic and thermodynamic measurements for free trypsinogen, as compared to its binary adduct with Ile-Val, can be ascribed to the substantial differences in the two molecular species, which include the spatial orientation of Asp189.

Animals↗
PubMed1984

Benzamidine as a spectroscopic probe for the primary specificity subsite of trypsin-like serine proteinases. A case for BPTI binding to bovine beta-trypsin.

Formation and dissociation of the benzamidine:beta-trypsin adduct is accompanied by reversible spectral changes in the ultraviolet region (between 230 and 300 nm). The pH-independent difference extinction coefficient of the adduct (benzamidine:beta-trypsin complex minus the free proteinase) is 1.75 mM-1 cm-1 at 248 nm. This signal can be used in studies of inhibitor and substrate binding by rapid kinetic techniques. Therefore, following the spectral changes associated with the displacement of benzamidine from the primary specificity subsite, the kinetics of the beta-trypsin:BPTI complex formation were investigated between pH 2.9 and 7.6 (I = 0.1 M) at 21 +/- 0.5 degree C. Under all the experimental conditions the beta-trypsin:BPTI complex formation, examined by benzamidine displacement experiments, may be described in terms of a simple competition event. On the other hand, the very same reaction followed by displacement of another spectroscopic probe, proflavine, appears to involve the ternary proflavine:beta-trypsin:BPTI adduct (7). The difference between the kinetic processes of beta-trypsin:BPTI complex formation, observed by using benzamidine and proflavine as reaction indicators, suggests that the two dye molecules bind at non-coincident regions of the proteinase active center. The advantages in using benzamidine as a sensitive probe specific for the S1 subsite of the recognition center of trypsin-like proteinases, as compared to proflavine, are emphasized.

Amidines↗
PubMed1984

Aromatic tetra-amidines: synthesis of halo-derivatives and their antiproteolytic activity.

Mono-halo derivatives of 1,3-di-(p-amidinophenoxy)-2,2-bis-(p-amidinophenoxymethyl)p ropane (TAPP-H) have been synthesized and their inhibitory effect on the bovine trypsin, bovine thrombin and porcine pancreatic kallikrein catalyzed hydrolysis of p-nitroanilides of amino acids was investigated, at pH 8.1 and 37 degrees, in parallel with that of TAPP-H and benzamidine. The addition of a halogen (Cl, Br or I) at position 2 of each benzamidine moiety of TAPP-H is accompanied by an increase of the inhibitory effect. This is especially evident in the case of porcine pancreatic kallikrein inhibition by TAPP-Cl. The structural basis of the different inhibitory effect of TAPP-H, TAPP-halo derivatives and benzamidine on the catalyzed hydrolysis of the proteinases examined are discussed and the role of Asp-189 in bovine trypsin, Asp-189 and Glu-149 or Asp-192 in bovine thrombin and Asp-189 and Asp-A148 or Glu-150 in porcine pancreatic kallikrein is focused.

Amidines↗
PubMed1983

Transient removal of proflavine inhibition of bovine beta-trypsin by the bovine basic pancreatic trypsin inhibitor (Kunitz). A case for "chronosteric effects".

The formation of the bovine beta-trypsin-bovine basic pancreatic trypsin inhibitor (Kunitz) (BPTI) complex was monitored, making use of three different signals: proflavine displacement, optical density changes in the ultraviolet region, and the loss of the catalytic activity. The rates of the reactions indicated by the three different signals were similar at neutral pH, but diverged at low pH. At pH 3.50, proflavine displacement precedes the optical density changes in the ultraviolet and the loss of enzyme activity by several orders of magnitude in time (Antonini, E., Ascenzi, P., Menegatti, E., and Guarneri, M. (1983) Biopolymers 22, 363-375). These data indicated that the bovine beta-trypsin-BPTI complex formation is a multistage process and led to the prediction that, at pH 3.50, BPTI addition to the bovine beta-trypsin-proflavine complex would remove proflavine inhibition and the enzyme would recover transiently its catalytic activity before being irreversibly inhibited by completion of BPTI binding. The kinetic evidences, by completion of BPTI binding. The kinetic evidences, here shown, verified this prediction, indicating that during the bovine beta-trypsin-BPTI complex formation one transient intermediate occurs, which is not able to bind proflavine but may bind and hydrolyze the substrate. Thus, the observed peculiar catalytic behavior is in line with the proposed reaction mechanism for the bovine beta-trypsin-BPTI complex formation, which postulates a sequence of distinct polar and apolar interactions at the contact area.

Acridines↗
PubMed1983

Catalytic properties of porcine pancreatic elastase: a steady-state and pre-steady-state study.

Pre-steady-state and steady-state kinetics for the p.p. elastase-catalysed hydrolysis of ZAlaONp, one of the most favourable substrates for this serine protease, have been studied between pH 4.0 and 8.0. The results are consistent with the minimum three-step mechanism: (formula; see text) Under pre-steady-state conditions, where [E0] much greater than [S0], the values of the dissociation constant of the E X S complex (Ks = k-1/k+1) and of the individual rate constants for the catalytic steps (k+2 and k+3) have been determined over the whole pH range explored. Under steady-state conditions, where [S0] much greater than [E0], the values of kcat and Km have been obtained over the same pH range. The pH profiles of k+2, k+3, k+2/Ks, kcat, kcat/Km reflect the ionization of a group, probably His57, with a pKa value of 6.85 +/- 0.10. The values of Ks and Km are pH independent. The steady-state parameters for the p.p. elastase-catalysed hydrolysis of a number of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids have been also determined between pH 4.0 and 8.0 and compared with those of b.beta-trypsin and b.alpha-chymotrypsin. For all the substrates examined the acylation step (k+2) is rate limiting in the p.p. elastase catalysis, between pH 4.0 and 8.0. The different catalytic behaviours of p.p. elastase, b.beta-trypsin and b.alpha-chymotrypsin are consistent with the known three-dimensional structures of these serine proteases.

Animals↗
PubMed1983

Structural investigations on pharmaceutical enzymes. Ornithine transcarbamylase (OCT) from bovine liver.

Ornithine transcarbamylase (OCT) is one of the urea-cycle enzymes in mitochondria which is involved in metabolic disorders in man. Reye's syndrome, characterized by encephalopathy and fatty infiltration of the liver, is one of such diseases. During this syndrome a reduction of OCT specific activity was observed, probably due to increased proteolytic degradation or to alteration in the enzyme conformation. It has recently been found that OCT administration to patients with liver disorders, contributes to illness resolution. Since the therapeutic properties of the enzymes are usually related to their structure, this paper reports some findings obtained by spectroscopic measurements on OCT structure and conformation stability in solution.

Animals↗
PubMed1982

Isolation and characterization of a new form of the porcine pancreatic secretory trypsin inhibitor. Biochemical studies and high-resolution 1H-NMR.

A new active form of porcine PSTI (pancreatic secretory trypsin inhibitor) was isolated during the fractionation by ion-exchange chromatography of the already known forms PSTI I and II. Biochemical and 1H-NMR techniques were used to characterize the new inhibitor, which is referred to as PSTI III. The amino acid composition, the nature of the N-terminal residue and data obtained from the tryptic peptides and indicate that PSTI III lacks the N-terminal octapeptide of PSTI I; hence, it starts and ends with disulfide bridges. The conclusion is supported by the 1H-NMR spectrum of the protein at 270 MHz. The biological activity and the most prominent conformational and dynamic features of forms I and II are retained in inhibitor III. However, PSTI III appears to be less compact than its parent forms I and II, suggesting that in the latter inhibitors an interaction between the N-terminal tail and the bulk of the protein may contribute to the overall stability. The genetic origin of PSTI III is discussed.

Amino Acid Sequence↗
PubMed1982

1H-NMR studies of the structure and stability of the bovine pancreatic secretory trypsin inhibitor.

The tertiary structure of the isoinhibitor A (Kazal) isolated from bovine pancreatic tissue has been characterized by 1H-NMR studies in 2H2O solution. A number of slowly exchanging backbone amides are observed. The dynamics of the aromatic side chains and their interaction with methyl groups from aliphatic residues are essentially identical in the bovine and in the homologous inhibitor from porcine pancreas. Both inhibitors contain two tyrosines in positions 20 and 31 as the only aromatic residues. The previous assignment of the aromatic resonances in the porcine inhibitor is valid for the bovine homolog, as deduced from selective nuclear Overhauser enhancement (NOE) experiments. The tyrosine which inhibits an NMR spectral pattern characteristic of rapid ring motion does not show appreciable NOE to backbone or sidechain protons, in agreement with the solvent exposure predicted for Tyr-20 in the porcine inhibitor. In contrast, the immobilized Tyr-31 exhibits a NOE pattern that indicates close interaction with a number of backbone amide protons and aliphatic side chain groups, confirming that the aromatic ring is buried. At 90 degrees C, pH 5, the protein is still substantially folded, as manifested by both backbone and side chain resonances. The results are compared with previous optical rotatory dispersion findings. A number of side chain resonances were assigned to specific amino acid residues in the sequence.

Amino Acid Sequence↗
PubMed1982

Catalytic properties of human urinary kallikrein.

Kinetic studies have been carried out with a well-characterized preparation of human urinary (h.u.) kallikrein using chromogenic substrates. Steady-state and pre-steady-state data for h.u. kallikrein catalyzed hydrolysis of N alpha-carbobenzoxy-L-lysine p-nitrophenyl ester (ZLysONp) and of N alpha-carbobenzoxy-L-alanine p-nitrophenyl ester (ZAlaONp) in the presence and absence of ethylamine and acetamidine have been obtained under various conditions and have been analyzed in the framework of the minimum three-step mechanism: (formula see text) The pH dependencies of the kinetic parameters for the hydrolysis of ZLysONp and ZAlaONp in the presence of saturating levels of ethylamine and acetamidine show that at acid pH values (less than or equal to 4) the k3 step (deacylation) is rate limiting in catalysis, whereas for pH values greater than or equal to 6, k2 (acylation) becomes rate limiting. On the other hand, the acylation step is rate limiting in the enzymatic hydrolysis of ZAlaONp over the whole pH range explored. Saturating concentrations of acetamidine increase, more than those of ethylamine, kcat for the hydrolysis of ZAlaONp. The affinity of h.u. kallikrein for acetamidine and ethylamine changes about 5-fold with pH between pH 5 and 3. The pH dependence of the spectral properties of free hu. kallikrein reflects the ionization of a group with a pKa value of 4.45 +/- 0.1. The results point out that, similarly to bovine beta-trypsin, h.u. kallikrein catalysis involves an ionizable group which has a pKa of about 4.5 in the free enzyme and a pKa of about 3.7 in the enzyme bound to cationic substrates or ligands.

Amidines↗