Substrate specificity of penicillin acylase from Streptomyces lavendulae

The kinetic parameters of several substrates of penicillin acylase from Streptomyces lavendulae have been determined. The enzyme hydrolyses phenoxymethyl penicillin (penicillin V) and other penicillins with aliphatic acyl-chains such as penicillin F, dihydroF, and K. The best substrate was penicillin K (octanoyl penicillin) with a k(cat)/K(m) of 165.3 mM(-1) s(-1). The enzyme hydrolyses also chromogenic substrates as NIPOAB (2-nitro-5-phenoxyacetamido benzoic acid), NIHAB (2-nitro-5-hexanoylamido benzoic acid) or NIOAB (2-nitro-5-octanoylamido benzoic acid), however failed to hydrolyse phenylacetil penicillin (penicillin G) or NIPAB (2-nitro-5-phenylacetamido benzoic acid) and penicillins with polar substituents in the acyl moiety. These results suggest that the structure of the acyl moiety of the substrate is more determinant than the amino moiety for enzyme specificity. The enzyme was inhibited by several organic acids and the extent of inhibition changed with the hydrophobicity of the acid. The best inhibitor was octanoic acid with a K(i) of 0.8 mM. All the results, taking together, point to an active site highly hydrophobic for this penicillin acylase from Streptomyces lavendulae.     

Chromogenic analogues of penicillin dihydroF and penicillin K for the continuous spectrophotometric determination of aliphatic penicillin acylase activity

The synthesis of 2-nitro-5-[(hexanoyl)-amino]-benzoic acid and 2-nitro-5-[(octanoyl)-amino]-benzoic acid as chromogenic substrates for the determination of aliphatic penicillin acylase activity is described. During enzymatic hydrolysis, the released chromophore, 2-nitro-5-amino-benzoic acid, was detected at 405 nm. Penicillin acylase from Streptomyces lavendulae had an appreciable activity towards these substrates, which can then be used to detect penicillin acylases able to cleave hexanoyl and octanoyl residues off synthetic amides as well as penicillin dihydroF and penicillin K, their natural analogues.     

A new colorimetric assay for methionyl aminopeptidases: examination of the binding of a new class of pseudopeptide analog inhibitors

A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (L-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV-Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using L-Met-p-NA, the kinetic constants k(cat) and K(m) were determined for EcMetAP-I and PfMetAP-II and were compared with those obtained with a "standard" high-performance liquid chromatography (HPLC) discontinuous assay. The assay has also been used to determine the temperature dependence of the kinetic constant k(cat) for PfMetAP-II as well as to screen two novel pseudopeptide inhibitors of MetAPs. The results demonstrate that L-Met-p-NA provides a fast, convenient, and effective substrate for both type I and type II MetAPs and that this substrate can be used to quickly screen inhibitors of MetAPs.     

Kinetic characteristics and enantioselective action of penicillinase in the hydrolysis reaction of N-phenylacetyl derivatives of 1-aminoethylphosphonic acid and its esters

Penicillin acylase from E. coli (EC 3.5.1.11) was found to hydrolyze N-phenylacetylated 1-aminoethylphosphonic acid and its esters. The enzyme preferentially converts the R-form of the substrates: the ratios of the bimolecular rate constants of penicillin acylasecatalyzed hydrolysis of R- and S-forms of 1-(N-phenylacetamino)-ethylphosphonic acid and its dimethyl- and diisopropyl-esters are 58000, 2300, 1800; these derivatives were shown to have the greatest values of the catalytic constants for enzymatic hydrolysis of all known substrates for penicillin acylase: 237, 148 and 134 s-1; the corresponding Km values are 3.7 10(-5), 6.8 10(-4) and 6.2 10(-4) M at pH 7.0. The kinetics of enzymatic hydrolysis of 1-(N-phenylacetamino)-ethylphosphonic acid was investigated up to high degrees of conversion. The inhibition of penicillin acylase by high concentrations of the R-form of the substrate (with substrate inhibition constant of 0.07 M) and competitive inhibition by the reaction product, phenylacetic acid (Ki = 3.5 10(-5) M), was observed.     

一株产多种β-内酰胺类抗生素酰化酶菌株的筛选

为了从大量的候选菌株中快速筛选头孢菌素酰化酶产生菌,设计并合成了一系列头孢菌素酰化酶的底物类似物。这些酰胺类的底物类似物由二部分组成,一部分为与头孢菌素相同或相似的侧链,另外一部分为发色基团或便于检测的基团。它们被酰化酶水解酰胺键以后可以方便快速的检测,因此用于对大量菌株进行快速筛选。采用这些化合物筛选到6株酰化酶阳性菌株。其中菌株ZH0650能够同时水解GL-7ACA和多个底物类似物。进一步研究表明,该菌至少产生3种酰化酶,AD-NABA酰化酶,青霉素G酰化酶和头孢菌素C酰化酶。我们初步纯化了AD-NABA酰化酶和青霉素G酰化酶,并对头孢菌素C酰化酶的活力进行了鉴定。这是首次报道的可以产生青霉素G酰化酶和头孢菌素酰化酶等多种酰化酶的菌株,具有良好的应用前景。     

Kinetics of enzyme acylation and deacylation in the penicillin acylase-catalyzed synthesis of beta-lactam antibiotics.

Penicillin acylase catalyses the hydrolysis and synthesis of semisynthetic beta-lactam antibiotics via formation of a covalent acyl-enzyme intermediate. The kinetic and mechanistic aspects of these reactions were studied. Stopped-flow experiments with the penicillin and ampicillin analogues 2-nitro-5-phenylacetoxy-benzoic acid (NIPAOB) and d-2-nitro-5-[(phenylglycyl)amino]-benzoic acid (NIPGB) showed that the rate-limiting step in the conversion of penicillin G and ampicillin is the formation of the acyl-enzyme. The phenylacetyl- and phenylglycyl-enzymes are hydrolysed with rate constants of at least 1000 s-1 and 75 s-1, respectively. A normal solvent deuterium kinetic isotope effect (KIE) of 2 on the hydrolysis of 2-nitro-5-[(phenylacetyl)amino]-benzoic acid (NIPAB), NIPGB and NIPAOB indicated that the formation of the acyl-enzyme proceeds via a general acid-base mechanism. In agreement with such a mechanism, the proton inventory of the kcat for NIPAB showed that one proton, with a fractionation factor of 0.5, is transferred in the transition state of the rate-limiting step. The overall KIE of 2 for the kcat of NIPAOB resulted from an inverse isotope effect at low concentrations of D2O, which is overridden by a large normal isotope effect at large molar fractions of D2O. Rate measurements in the presence of glycerol indicated that the inverse isotope effect originated from the higher viscosity of D2O compared to H2O. Deacylation of the acyl-enzyme was studied by nucleophile competition and inhibition experiments. The beta-lactam compound 7-aminodesacetoxycephalosporanic acid (7-ADCA) was a better nucleophile than 6-aminopenicillanic acid, caused by a higher affinity of the enzyme for 7-ADCA and complete suppression of hydrolysis of the acyl-enzyme upon binding of 7-ADCA. By combining the results of the steady-state, presteady state and nucleophile binding experiments, values for the relevant kinetic constants for the synthesis and hydrolysis of beta-lactam antibiotics were obtained.     

Integral kinetics of alpha-galactosidase purified from Glycine max for simultaneous hydrolysis of stachyose and raffinose

alpha-Galactosidase from soybean (Glycine max) was purified by a five-step procedure. The enzyme's natural substrates, raffinose and stachyose, have K(m)'s of 3. 0 mM and 4. 79 mM, respectively. The products, galactose and sucrose, were measured after separation by liquid chromatography. Galactose is a competitive product inhibitor of stachyose and raffinose hydrolysis with a K(i) of 0. 12 mM. We determined these parameters by an integral kinetic approach. Stachyose hydrolysis gives a nearly constant level of raffinose shortly after hydrolysis begins. Thus, cleavage of the first alpha-(1,6)-bond in the tetrasaccharide is the rate-limiting step. Since the stachyose hydrolysis yields raffinose, soybean alpha-galactosidase simultaneously hydrolyzes two substrates. We present a novel approach for analyzing simultaneous substrate hydrolysis with competitive product inhibition by a modified integral rate expression. The experimentally found kinetic parameters are confirmed by solving the simultaneous equations which describe the hydrolysis. This technique may be applicable to other hydrolytic enzymes with multiple substrates.     

A new method for spectrophotometric assay of activity of cross-linked penicillin acylase aggregates

A new method for monitoring reactions catalyzed by an immobilized enzyme, cross-linked penicillin acylase aggregates (PA CLEA), is suggested. Appropriate chromogenic substrates for spectrophotometric assay of catalytic activity of immobilized enzyme were chosen and their kinetic parameters determined. Active sites in PA CLEA preparations were titrated by the suggested method; it is shown that almost all active sites are retained during immobilization. This method is characterized as highly expressive, simple, and precise and may be used for control of PA immobilization efficiency as well as for study of operational, thermal, and pH stability of immobilized enzyme preparations.     

Assay for rapid analysis of the tri-peptidase activity of LTA4 hydrolase

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme with an epoxide hydrolase activity as well as an arginyl tri-peptidase activity. Detailed enzymological and mechanistic investigations of the latter activity have been hampered by the lack of a rapid and convenient enzyme assay. Here we have developed a new method allowing direct spectrophotometric assessment of the tri-peptide cleaving activity of leukotriene A4 hydrolase, as well as other peptidases. The method utilizes two competing substrates, one chromogenic reference substrate together with the tri-peptide substrate of interest, and relies on computer-assisted analysis of progress curves. The chromogenic reference substrate serves to disclose the "invisible" tri-peptide substrate for kinetic analysis. The method is fast and simple and will allow detailed kinetic studies and screening for natural peptide substrates of leukotriene A4 hydrolase as well as other members of the M1 family of aminopeptidases.     

Enzymatic reaction of silent substrates: kinetic theory and application to the serine protease chymotrypsin

Investigating the selectivity that an enzyme expresses toward its substrates can be technically challenging if reaction of these substrates is not accompanied by a conveniently monitored change in some physicochemical property. In this paper, we describe a simple method for determining steady-state kinetic parameters for enzymatic turnover of such "silent" substrates. According to this method, silent substrate S is allowed to compete for enzymic reaction with signal-generating substrate S*, whose conversion to product can be conveniently monitored. Full reaction progress curves are collected under conditions of [S*](o) < K(m)* and [S](o) >or= 3K(m). Progress curves collected under these conditions are characterized by an initial lag phase of duration tau that is followed by the pseudo-first-order reaction of S. Steady-state kinetic parameters for the silent substrate can be obtained by one of two methods. One method combines least-squares fitting with numerical integration of appropriate rate equations to analyze the progress curves, while the other method relies on direct graphical analysis in which K(m) is the value of [S](o) that reduces the control velocity by a factor of 2 and V(max) is shown to simply equal the ratio [S](o)/tau. We use these methods to analyze the alpha-chymotrypsin-catalyzed hydrolysis of silent substrate Suc-Ala-Phe-AlaNH(2) with signal generator Suc-Ala-Phe-pNA. From the curve-fitting method, k(c) = 0.9 +/- 0.2 s(-1) and K(m) = 0.4 +/- 0.1 mM, while by direct graphical analysis, k(c) = 1.1 +/- 0.1 s(-1) and K(m) = 0.51 +/- 0.03 mM. As validation of this new method, we show agreement of these values with those determined independently by HPLC analysis of the hydrolysis of Suc-Ala-Phe-AlaNH(2) by alpha-CT, where k(c) = 1.1 +/- 0.1 s(-1) and K(m) = 0.5 +/- 0.1 mM.     

A kinetic expression for hydrolysis of soluble starch by glucoamylase

As the hydrolysis of starch by glucoamylase proceeds with stepwise removal of glucose units from the nonreducing ends of the starch chain, the number of available substrate molecules is essentially unchanged in the course of the degradation. In view of this aspect, a simple practical kinetic expression, which consists of a modified Michaelis-Menten form with product inhibition, is presented for the hydrolysis of soluble starch. It is assumed that the values of kinetic parameters V(m) and K(m) vary linearly from the values for starch toward those for maltose. The applicability of this kinetic expression is verified through the simulation with the experimental results for the hydrolysis of two soluble starches with different average molecular weights of 3 x 10(4) and 3 x 10(6).     

Effect of Calcium Ions on Enteropeptidase Catalysis

The effects of calcium ions on hydrolysis of low molecular weight substrates catalyzed by different forms of enteropeptidase were studied. A method for determining activity of truncated enteropeptidase preparations lacking a secondary trypsinogen binding site and displaying low activity towards trypsinogen was developed using N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25 degrees C were determined for natural enteropeptidase (K(m) 59.6 microM, k(cat) 6660 min(-1), k(cat)/K(m) 111 microM(-1) x min(-1)), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (K(m) 176.9 microM, k(cat) 6694 min(-1), k(cat)/K(m) 37.84 microM(-1) x min(-1)) and trypsin (K(m) 56.0 microM, k(cat) 8280 min(-1), k(cat)/K(m) 147.86 microM(-1) x min(-1)). It was shown that the enzymes with trypsin-like primary active site display similar hydrolysis efficiency towards Z-Lys-S-Bzl. Calcium ions cause 3-fold activation of hydrolysis of the substrates of general type GD(4)K-X by the natural full-length enteropeptidase. In contrast, the hydrolysis of substrates with one or two Asp/Glu residues at P2-P3 positions is slightly inhibited by Ca2+. In the case of enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 fragment), the activating effect of calcium ions was not detected for all the studied substrates. The results of hydrolysis experiments with synthetic enteropeptidase substrates GD(4)K-F(NO(2))G, G(5)DK-F(NO(2))G (where F(NO(2)) is p-nitrophenyl-L-phenylalanine residue), and GD(4)K-Nfa (where Nfa is beta-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.     

Penicillin amidohydrolases in fungal autolysis

The production of penicillin G and penicillin V amidohydrolases or acylases (E.C.3.5.1.11) was studied during the autolysis of filamentous fungi in a mineral medium, and in the same medium with phenoxyacetic acid as inducer. In all the studied fungi, enzymes showing penicillin G and penicillin V amidohydrolase activities were found. Generally, an increase of these activities during fungal autolysis was observed. The presence of phenoxyacetic acid in the medium did not increase these activities. The activities found in the culture fluids were generally higher than that found in the mycelial extracts. Under these conditions, beta-lactamases (penicillinases) were not found. The fungi Alternaria alternata, Fusarium culmorum, Penicillium oxalicum, and the species Penicillium 222 were chosen to study penicillin G and penicillin V acylases. The enzymes were precipitated with tannic acid from the culture fluid of their autolyzed cultures. Some kinetic constants of these activities were determined.     

Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Leishmania major

Kinetic properties of the dimeric enzyme dUTPase from Leishmania major were studied using a continuous spectrophotometric method. dUTP was the natural substrate and dUMP and PPi the products of the hydrolysis. The trypanosomatid enzyme exhibited a low K(m) value for dUTP (2.11 microM), a k(cat) of 49 s(-1), strict Michaelis-Menten kinetics and is a potent catalyst of dUDP hydrolysis, whereas in other dUTPases described, this compound acts as a competitive inhibitor. Discrimination is achieved for the base and sugar moiety showing specificity constants for different dNTPs similar to those of bacterial, viral, and human enzymes. In the alkaline range, the K(m) for dUTP increases with the dissociation of ionizable groups showing pK(a) values of 8.8, identified as the uracil moiety of dUTP and 10, whereas in the acidic range, K(m) is regulated by an enzyme residue exhibiting a pK(a) of 7.1. Activity is strongly inhibited by the nucleoside triphosphate analog alpha-beta-imido-dUTP, indicating that the enzyme can bind triphosphate analogs. The existence of specific inhibition and the apparent structural and kinetic differences (reflected in different binding strength of dNTPs) with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against leishmaniasis.     

A spectrophotometric assay for determining the rate constants of acetylcholinesterase inhibitions.

A spectrophotometric assay procedure has been developed for determining the rate constants for the inhibition of acetylcholinesterase by carbamates and phosphates. The method permits the investigation of inhibitors over a large range in the value of the phosphorylation or carbamylation rate constants without involving the kinetic parameters of the assay substrate in the calculations.     

Assay of penicillin acylase in organic media

Summary The synthetic substrate 6-nitro-3-(phenylacetamido) benzoic acid (NIPAB) is an appropriate substrate for assaying penicillin acylase activity in reversed micellar systems of Aerosol - OT in isooctane. Accumulation of 6-nitro-3-aminobenzoic acid (NABA) produced by the enzymatic hydrolysis of NIPAB, followed by the increase in absorbance at 405 nm, was linear at 4 to 20 mM for up to 30 minutes and 15 °C to 40 °C.Abbreviations PA penicillin acylase (penicillin amidohydrolase EC 3.5.1.11) - AOT Aerosol OT (sodium bis- (2-ethylhexyl) sulfosuccinate) - NIPAB 6-nitro-3-(phenylacetamido)-benzoic acid - NABA 6-nitro-3-aminobenzoic acid - BSA bovine serum albumin     

Enzymatic hydrolysis of cellulosic materials: A kinetic study

A kinetic study of the enzymatic hydrolysis of two celluloses with different structural features was performed at various temperatures (26-50 degrees C). The enzymatic system consisted of three types of enzymes: E(1)-beta-1,4-glucan glucanohydrolase; E(2)-beta-1,4-glucan cellobiohydrolase; and E(3)-beta-glucosidase. A mathematical model for the mechanism of the hydrolysis of cellulosic materials catalyzed by a multienzymatic system was checked and a good rationalization of the experimental results was achieved. Uncompetitive and competitive glucose inhibition on E(1) and E(2), respectively, appeared to occur for both substrates. Inhibition by cellobiose was checked at 34 degrees C on one substrate. The V(max), K(m), and glucose inhibition constants were optimized and their dependence on temperature determined.     

Kinetics and energetics of ligand binding determined by microcalorimetry: insights into active site mobility in a psychrophilic alpha-amylase

A new microcalorimetric method for recording the kinetic parameters k(cat), K(m) and K(i) of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic alpha-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic alpha-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic alpha-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site.     

Interaction kinetics of reversible inhibitors and substrates with acetylcholinesterase and its fasciculin 2 complex

Fasciculin 2 (Fas2), a three-fingered peptide of 61 amino acids, binds tightly to the peripheral site of acetylcholinesterases (AChE; EC ), occluding the entry portal into the active center gorge of the enzyme and inhibiting its catalytic activity. We investigated the mechanism of Fas2 inhibition by studying hydrolysis of cationic and neutral substrates and by determining the kinetics of interaction for fast equilibrating cationic and neutral reversible inhibitors with the AChE.Fas2 complex and free AChE. Catalytic parameters, derived by eliminating residual Fas2-resistant activity, reveal that Fas2 reduces k(cat)/K(m) up to 10(6)-fold for cationic substrates and less than 10(3)-fold for neutral substrates. Rate constants for association of reversible inhibitors with the active center of the AChE.Fas2 complex were reduced about 10(4)-fold for both cationic and neutral inhibitors, while dissociation rate constants were reduced 10(2)-to 10(3)-fold, compared with AChE alone. Rates of ligand association with both AChE and AChE.Fas2 complex were dependent on the protonation state of ionizable ligands but were also markedly reduced by protonation of enzyme residue(s) with pK(a) of 6.1-6.2. Linear free energy relationships between the equilibrium constant and the kinetic constants show that Fas2, presumably through an allosteric influence, markedly alters the position of the transition state in the reaction pathway. Since Fas2 complexation introduces an energetic barrier for hydrolysis of substrates that exceeds that found for association of reversible ligands, Fas2 influences catalytic parameters by a more complex mechanism than simple restriction of diffusional entry and exit from the active center. Conformational flexibility appears critical for facilitating ligand passage in the narrow active center gorge for both AChE and the AChE.Fas2 complex.     

Two continuous spectrophotometric assays for methionine aminopeptidase

Two spectrophotometric assays have been developed for methionine aminopeptidases (MetAPs). The first method employs a thioester substrate which, upon enzymatic removal of the N-terminal methionine, generates a free thiol group. The released thiol is quantitated using Ellman's reagent. The MetAP reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion, with the addition of an excess of Ellman's reagent into the assay reaction. Two tripeptide analogues were synthesized and found to be excellent substrates of both Escherichia coli MetAP and human MetAP2 (k(cat)/K(M) = 2.8 x 10(5) M(-1) s(-1) for the most reactive substrate). In the second assay method, the MetAP reaction is coupled to a prolyl aminopeptidase reaction using Met-Pro-p-nitroanilide as substrate. MetAP-catalyzed cleavage of the N-terminal methionine produces prolyl-p-nitroanilide, which is rapidly hydrolyzed by the prolyl aminopeptidase from Bacillus coagulans to release a chromogenic product, p-nitroaniline. This allows the MetAP reaction to be continuously monitored at 405 nm on a UV-VIS spectrophotometer. The assays have been applied to determine the pH optima and kinetic constants for the E. coli and human MetAPs as well as to screen MetAP inhibitors. These results demonstrate that the current assays are convenient, rapid, and sensitive methods for kinetic studies of MetAPs and effective tools for screening MetAP inhibitors.