Hydrolysis Of P-nitroanilide Research Articles

Effect of Human Serum Albumin on the Kinetics of N-glutaryl-L-phenylalanine p-nitroanilide Hydrolysis Catalyzed by α-Chymotrypsin

The effect of human serum albumin (HSA) addition on the rate of hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) catalyzed by α-chymotrypsin has been measured in phosphate buffer saline at pH=7.4. The presence of HSA (up to 200μM) leads to a decrease in the rate of the process. The reaction follows a Michaelis-Menten mechanism under all the conditions employed. To take into account the effect of substrate depletion due to its binding to albumin ultrafiltration experiments were carried out from which the binding of GPNA to HSA was derived. After correction of the kinetic data taking into account the binding of GPNA to HSA, the activity of the enzyme, and the derived Michaelis constant and catalytic rate constant tends to remain almost independent of the presence of albumin, indicating that the depletion of the substrate due to its binding to HSA is the main factor affecting the enzyme activity.

Kinetics of N-glutaryl- l-phenylalanine p-nitroanilide hydrolysis catalyzed by α-chymotrypsin in aqueous solutions of alkyltrimethylammonium bromides

The rate of N-glutaryl- l-phenylalanine p-nitroanilide hydrolysis catalyzed by α-chymotripsin has been measured in aqueous solutions of cetyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and dodecyltrimethylammonium bromide at concentrations below and above their critical micellar concentrations (CMC). For the three surfactants considered superactivity was observed, with maximum catalytic efficiencies taking place near the corresponding CMCs. The effect of the surfactants after the CMCs is mostly due to a decreased thermodynamic activity of the substrate due to its incorporation into the micelles. After addition of the surfactants, the Michaelis constant values (corrected to take into account the free substrate concentration) tend to decrease, passing through an ill defined minimum, afterwards reaching a constant value. The catalytic rate constants show the same profiles that the catalytic efficiency, being maxima near the surfactants CMCs. This maximum is more important for the surfactant having the shorter tail. This result is explained by considering that the hydrophobicity of the surfactant influences more the CMC than its association to the enzyme.

Discovery of a Dual-Function Peptide That Combines Aminopeptidase N Inhibition and Kinin B1Receptor Antagonism

Previous analyses support that aminopeptidase N is a major inactivation pathway for high-affinity peptide ligands of the human and rabbit forms of the kinin B(1) receptor (agonists or antagonists). In this study, we found that the high-affinity antagonist B-9958 (Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; des-Arg(9)-BK, des-arginine(9)-bradykinin) is an aminopeptidase N substrate based on its capacity to compete for the hydrolysis of the chromogenic substrate L-Ala-p-nitroanilide by membranes isolated from human or rabbit arterial smooth muscle cells, its inactivation in the presence of these membranes (radioreceptor assay) and on its intense potentiation by the aminopeptidase N inhibitor amastatin in the rabbit aorta contractility assay (gain of 0.84 units in the pA(2) scale). Analogs of B-9958 in which the N-terminal Lys residue was substituted by D-Lys or D-Arg (B-10352 and B-10356, respectively) showed reduced affinity at the human or rabbit B(1) receptors (1.2-2.8-fold), as estimated by the displacement of [(3)H]Lys-des-Arg(9)-BK binding, but were more potent antagonists of des-Arg(9)-BK-induced contraction of the rabbit aorta than B-9958 in the absence of amastatin; they were not potentiated by the latter inhibitor. Unexpectedly, B-10356 inhibited L-Ala- p-nitroanilide hydrolysis without being inactivated, suggesting that it is an aminopeptidase N inhibitor. This was verified because B-10356 (but not B-10352) potentiated peptides unrelated to kinins but susceptible to aminopeptidase N inactivation (angiotensin III, thrombin receptor hexapeptide agonist). B-10356 inhibits dual molecular targets (aminopeptidase N enzyme K(i), 0.9-2.2 microM; kinin B(1) receptor binding K(i), 0.5-1.5 nM), and this may be an advantage for specific therapeutic applications (e.g., inhibition of angiogenesis).

Kinetics of N-glutaryl- L-phenylalanine p-nitroanilide hydrolysis catalyzed by α-chymotrypsin in aqueous solutions of dodecyltrimethylammonium bromide

The rate of hydrolysis of N-glutaryl- L-phenylalanine p-nitroanilide (GPNA) catalyzed by α-chymotrypsin ( α-CT) has been measured in aqueous solutions of dodecyltrimethylammonium bromide (DTAB) at concentrations below and above the critical micelle concentration, as well as in the absence of surfactant. Under all the conditions employed, the reaction follows a Michaelis–Menten mechanism. The presence of the surfactant leads to superactivity below and above the critical micelle concentration (CMC), with a maximum reaction rate taking place near the CMC when the results are treated in terms of the analytical concentration of the substrate. A similar behavior was observed by working with the enzyme partially deactivated in the presence of 4 M urea. After correction to take into account the partitioning of the substrate between the micelles and the external media, the activity of the enzyme tends to remain almost constant above the corresponding CMCs. This results from a compensation of a decrease in the catalytic constant ( k cat ) and a decrease in the Michaelis constant ( K M ). The behavior of α-CT in the hydrolysis of GPNA in DTAB solutions is at variance with that previously reported for the hydrolysis of 2-naphthyl acetate in solutions of the same surfactant (E. Abuin, E. Lissi, R. Duarte, Langmuir 19 (2003) 5374). An explanation of the different effects of the surfactant on the behavior of the enzyme with both substrates is advanced, taking into account the complexity of the mechanism of the α-CT-mediated reaction, more specifically, in terms of different rate-limiting steps for the formation of the measured products.

Kinetic study on conformational effect in hydrolysis of p-nitroanilides catalyzed by α-chymotrypsin

Effects of medium viscosity on kinetics for the hydrolysis of p-nitroanilides of certain amino acid derivatives catalyzed by α-chymotrypsin have been investigated. Observed data indicate that the overall rate constant, kcat, is hardly affected by the medium viscosity in all the substrates employed and equals the rate constant at the acylation step, k2, in the measured range of viscosity. By comparison with the data on p-nitrophenyl ester substrates reported previously, it is concluded that the formation of the tetrahedral intermediate in the course of the acylation of the enzyme is influenced by conformational change of the enzyme, whereas the breakdown of the intermediate is almost free from conformational effects.

α-Chymotrypsin superactivity in aqueous solutions of cationic surfactants

α-Chymotrypsin (α-CT) activity was tested in aqueous media with the following cetyltrialkylammonium bromide surfactants in the series methyl, ethyl, propyl and butyl, different in the head group size, and for the sake of comparison also with the anionic sodium n-dodecyl sulfate and the zwitterionic myristyldimethylammonium propanesulfonate. N-glutaryl- l-phenylalanine p-nitroanilide hydrolysis rate was monitored at surfactant concentration above the critical micellar one. Only some cationic surfactants gave superactivity and the head group size had a major weight. The highest superactivity was measured in the presence of cetyltributylammonium bromide. The effect of both nature and concentration of three different buffers was also investigated. There is a dependence of enzyme superactivity on buffer type. Michaelis–Menten kinetics were found. The binding constants of substrate with micellar aggregates were determined in the used buffers and the effective improvement of reaction rate (at the same free substrate concentration in the medium) was calculated. k cat significantly increased while K m was little changed after correction to free substrate concentration. The ratio of k cat to K m was between 12 and 35 times higher than in pure buffer, depending on buffer and surfactant concentrations. The increase of α-CT activity (30%) was less important in the presence of 1×10 −2 M tetrabutylammonium bromide, a very hydrophobic salt, unable to micellise. Fluorescence spectra showed differences of enzyme conformation in the presence of various surfactants.

Two cell-wall-associated aminopeptidases from Lactobacillus helveticus and the purification and characterization of APII from strain ITGL1

Lactobacillus helveticus ITGL1 is able to hydrolyse many amino-acyl and dipeptidyl-p-nitroanilides. Analysis of heat inactivation kinetics, metal ion and protease inhibitor effects, and the subcellular location of aminopeptidase activities in both the parental strain and mutants deficient in lysyl-p-nitroanilide hydrolysis, led to the characterization of two cell-wall-associated aminopeptidases, APII and APIV. APII, which catalysed L-lysine p-nitroanilide hydrolysis, was purified about 28-fold to homogeneity from cell-wall extracts of L. helveticus ITGL1 and characterized. The purified enzyme appeared to be monomeric, with a molecular mass of 97 kDa. Aminopeptidase activity was greatest at pH 6.5 and 50 degrees C. APII was completely inhibited by bestatin, chelating agents such as EDTA or 1,10-phenanthroline and the divalent cations Zn2+ and Cu2+. The activity of the EDTA-treated enzyme was restored by Co2+, Ca2+ or Mn2+. Although APII was able to degrade several dipeptides and tripeptides with hydrophobic N-terminal amino acid (Leu, Ala), it was inactive on peptides containing Pro or Gly, and may thus contribute to the development of cheese flavour by processing bitter peptides.

A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins.

A homogeneous serine proteinase secreted by the extreme halophilic bacterium Halobacterium mediterranei 1538 was isolated by affinity chromatography on bacitracin-Sepharose with a yield of 48% (260-fold purification). The enzyme reveals an optimum for pyroglutamyl-Ala-Ala-Leu p-nitroanilide hydrolysis at pH 8.0-8.5 (Km 0.14 mM; k(cat). 36.9 s-1). Its activity increases linearly with NaCl concentration over the range 2-5 M. The substrate specificity of the enzyme is comparable with that of secretory subtilisins, the extent of protein degradation approaching that attained with proteinase K. The enzyme has a molecular mass of 41 kDa and a pI of 7.5. The N-terminal sequence of H. mediterranei serine proteinase reveals a 50% identity with that of Thermoactinomyces vulgaris serine proteinases, indicating that the enzyme belongs to the subtilisin family. Hence the serine proteinase secreted by the halophilic bacterium should be considered as a functional analogue, and a structural homologue, of eubacterial serine proteinases (subtilisins).

Catalysis by human leukocyte elastase: mechanistic insights into specificity requirements.

Steady-state kinetic parameters were determined for the human leukocyte elastase catalyzed hydrolysis of a series of peptide-based thiobenzyl esters and p-nitroanilides. The peptide units are MeOSuc-Val, MeOSuc-Alan-Pro-Val (n = 0-2), and MeOSuc-Alan-Pro-Ala (n = 1 or 2). The results of this study suggest five important mechanistic features for HLE. Few important remote subsite contacts are established in the Michaelis complex. Full recognition and tight binding of the substrate occurs in the transition state for acylation. The P3-S3 interaction is critical during acylation. Subsite contacts are unimportant in deacylation. P1 specificity is regulated by peptide length. An important steady-state kinetic consequence of this specificity is that the rate-limiting step of kc for p-nitroanilide hydrolysis changes from acylation to deacylation as the peptide chain is lengthened.

Purification and Characterization of Leu-Proteinase, the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves

The leucine specific serine proteinase present in the soluble fraction of leaves from Spinacia oleracea L. (called Leu-proteinase) has been purified by acetone precipitation and a combination of gel-filtration, ion exchange, and adsorption chromatography. This enzyme shows a molecular weight of 60,000 +/- 3,000 daltons, an isoelectric point of 4.8 +/- 0.1, and a relative electrophoretic mobility of 0.58 +/- 0.03. The Leu-proteinase catalyzed hydrolysis of p-nitroanilides of N-alpha-substituted(-l-)amino acids as well as of chromogenic macromolecular substrates has been investigated between pH 5 and 10 at 23 +/- 0.5 degrees C and I = 0.1 molar. The enzyme activity is characterized by a bell-shaped profile with an optimum pH value around 7.5, reflecting the acid-base equilibrium of groups with pK(a) values of 6.8 +/- 0.1 and 8.2 +/- 0.1 (possibly the histidyl residue present at the active site of the enzyme and the N-terminus group). Among the substrates considered, N-alpha-benzoyl-l-leucine p-nitroanilide shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 1 x 10(-9) molar. In agreement with the enzyme specificity, only N-alpha-tosyl-l-leucine chloromethyl ketone, di-isopropyl fluorophosphate and phenylmethylsulfonyl fluoride, among compounds considered specific for serine enzymes, strongly inhibit the Leu-proteinase. Accordingly, the enzyme activity is insensitive to cations, chelating agents, sulfydryl group reagents, and activators.

ChemInform Abstract: Ethyl Esters of N‐(3‐ and N‐(4‐Amidinobenzoyl) L‐Amino Acids: Synthesis and Antiproteolytic Activity Towards Bovine Trypsin and Porcine Pancreatic Kallikresin.

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.

ChemInform Abstract: AROMATIC TETRA‐AMIDINES: SYNTHESIS OF HALO‐DERIVATIVES AND THEIR ANTIPROTEOLYTIC ACTIVITY

AbstractPentaerythrit‐tetrabromid (I) reagiert mit den Phenolaten (II) zu Tetra‐ I phenoxy‐pentaerythriten (III), in denen die Cyano‐Gruppe in die Amidin‐Gruppe umgewandelt werden kann.

Fractionation of heparin by affinity chromatography on covalently-bound human α-thrombin

Commerical heparin, 135 USP units/mg, was fractionated by human α-thrombin-agarose affinity chromatography. Heparin was applied to an α-thrombin-agarose column equilibrated with 0.01 M Tris HCl (pH 7.4). Unbound heparin was washed from the column with the equilibration buffer. Bound heparin could be eluted with buffer containing 0.025 M NaCl. The specific activity of bound heparin was as great as 500 USP units/mg. Gel filtration was used to fractionate the heparin into molecular size classes. Low molecular weight heparin, with an average specific activity of 100 USP units/mg, was applied to the α-thrombin-agarose column. Gel filtration of the unbound heparin indicated that larger heparin molecules been selectively removed by the α-thrombin-agarose column. Bound heparin had a specific activity of 270 units/mg. Kinetic results of N-α-tosyl-L-glycyl-L-prolyl-L-arginine- p-nitroanilide hydrolysis by α-thrombin in the presence of heparin correlated with the anticoagulant activity.

Purification and properties of a periplasmic aminoendopeptidase from Escherichia coli.

A periplasmic aminoendopeptidase from Escherichia coli has been purified to hemogeneity. It is a monomer of molecular weight 45000 and containing one -- SH group that is necessary for catalytic activity. The study of its substrate specificity indicated that the enzyme has both aminopeptidase and endopeptidase activity. The pH optimum for L-alanine p-nitroanilide hydrolysis is between 7 and 7.5 and that for 125I-labeled casein proteolysis between 7.3 and 7.6. The activation energy for the hydrolysis of L-anine p-nitroanilide was calculated to be 5.3 kcal X mol-1 (22.2 kJ X mol-1).

Inhibitory Conditions of the Tryptic Hydrolysis of p-Nitroanilide by Colloidal Dispersion Formed in the Reaction Mixture

Inhibitory conditions of the tryptic hydrolyzing activity of benzoyl-DL-arginine p-nitro-anilide were investigated. Trypsin was inhibited probably according to its absorption on a colloidal dispersion which was formed in the reaction mixture containing zinc chelate of o-phenanthroline, the substrate, and phosphate of the buffer constituent over pH 6. Although zinc showed the same effect as its chelate compound did in the higher conc. of the substrate, the presence of more than 2 moles of o-phenanthroline to 1 mole of zinc prevented the formation of the colloidal dispersion. It was also found that skeleton and side chains of o-phenanthroline and its derivatives were influential factors to show inhibitory effects on the hydrolase activity of trypsin.

Studies on the mechanism of rat urinary kallikrein catalysis, and its relation to catalysis by trypsin

The pH-activity curves for rat urinary kallikrein catalysis of benzoyl- l-arginine ethyl ester and benzoyl- dl-arginine p-nitroanilide hydrolysis suggest the participation of a histidyl residue in the catalytic process. A specific active center reagent of trypsin, 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), did not inactivate the kallikrein at pH 7.2, 8.0, or 8.92. This was interpreted as the result of a fitting between TLCK and kallikrein, different from that between TLCK and trypsin, such that renders alkylation of a histidyl residue impossible. Phenylguanidine sulfate, cyclohexylguanidine sulfate, benzamidine-HCl, and β-naphthamidine-HCl were shown to competitively inhibit the kallikrein. The K i values found demonstrate differences between binding of the inhibitors to kallikrein and to trypsin, but assure the proposition of hydrophobic and anionic binding sites in the kallikrein active center. The data prove that trypsin and the trypsin-like kallikreins can be distinguished on the basis of the lack of reactivity of the latter toward TLCK.