Evidence for the involvement of arginyl residue at the active site of penicillin G acylase from Kluyvera citrophila

Penicillin G acylase (PGA) is used for the commercial production of semi-synthetic penicillins. It hydrolyses the amide bond in penicillin producing 6-aminopenicillanic acid and phenylacetate. 6-Aminopenicillanic acid, having the beta-lactam nucleus, is the parent compound for all semi-synthetic penicillins. Penicillin G acylase from Kluyvera citrophila was purified and chemically modified to identify the role of arginine in catalysis. Modification with 20 mM phenylglyoxal and 50 mM 2,3-butanedione resulted in 82% and 78% inactivation, respectively. Inactivation was prevented by protection with benzylpenicillin or phenylacetate at 50 mM. The reaction followed psuedo-first order kinetics and the inactivation kinetics (V(max), K(m), and k(cat)) of native and modified enzyme indicates the essentiality of arginyl residue in catalysis.     

Replacement of lysine 234 affects transition state stabilization in the active site of beta-lactamase TEM1

Lysine 234 is a residue highly conserved in all beta-lactamases, except in the carbenicillin-hydrolyzing enzymes, in which it is replaced by an arginine. Informational suppression has been used to create amino acid substitutions at this position in the broad spectrum Escherichia coli beta-lactamase TEM-1, in order to elucidate the role of this residue which lies on the wall at the closed end of the active site cavity. The mutants K234R and K234T were constructed and their kinetic constants measured. Replacement of lysine 234 by arginine yields an enzyme with similar activity toward cephalosporins and most penicillins, except toward the carboxypenicillins for which the presence of the guanidine group enhances the transition state binding. The removal of the basic group in the mutant K234T yields a protein variant which retains a low activity toward penicillins, but losts drastically its ability to hydrolyze cephalosporins. Moreover, these two mutations largely decreased the affinity of the enzyme for penicillins (10-fold for K234R and 50-fold for K234T). This can be correlated with the disruption of the predicted electrostatic binding between the C3 carboxylic group of penicillins and the amine function of the lysine. Therefore, lysine 234 in the E. coli beta-lactamase TEM-1 is involved both in the initial recognition of the substrate and in transition state stabilization.     

Separation by high-performance liquid chromatography of penicillins with C4 to C10 aliphatic side chains

A suitable and rapid method for the simultaneous determination of different aliphatic penicillins is described. Butyryl-, pentanoyl-, hexanoyl-, heptanoyl-, octanoyl-, nonanoyl- and decanoylpenicillin can be completely separated by high-performance liquid chromatography using an isocratic elution mode (50 mM H2KPO4, pH 5.0:methanol, 45:55 v/v). Under these conditions, retention times for C4 to C10 aliphatic side-chain penicillins were 2.5, 2.8, 4.1, 5.8, 8.9, 15.3, and 28.2 min. The benzylpenicillin retention time was 3.3 min.     

Detection and side-chain specificity of IgE antibodies to flucloxicillin in allergic subjects

Unlike studies on the antigenicity of penicillins in laboratory animals, limited information is available on the allergenicity of penicillins in man, especially with regard to fine structural allergenic differences between the many different penicillins. Inconsistent with the earlier conclusions of others, our studies suggest that side-chain structures to flucloxacillin-reactive IgE antibodies. Quantitative hapten inhibition studies revealed potent inhibition by flucloxacillin and three structurally related penicillins: oxacillin, cloxacillin and dicloxacillin. Analysis of the results showed that the side-chain group of flucloxacillin, 3-2(-chloro-6-fluorophenyl)-5-methyl-4-isoxazolyl, is recognized by some antibodies and that the 5-methyl-3-phenyl-4-isoxazolyl group, with or without halogen substituents, accounts for the reactivity of other antibodies and for the cross-reactions seen with some other penicillins. Since it is the side-chain group that distinguishes the many different types of penicillin, and since IgE antibodies in many of the allergic reactions recognize the side-chain groups, it is becoming clear that specific assays are required for the detection of IgE antibodies to each of the different penicillins.     

Development of analytical methods for some penicillins in bovine milk by ion-paired chromatography and confirmation by thermospray mass spectrometry

Analytical methods for the determination of cloxacillin, ampicillin/hetacillin, and amoxicillin in bovine milk were developed. The methods involved ultrafiltration of milk diluted with methanol, acetonitrile, and water on a 10 000-dalton cut-off filter. Separation of penicillins from other milk components was accomplished by ion-paired chromatography using a microbore column. The penicillins were detected using ultraviolet photodiode array (UV-PDA) detection and confirmed by thermospray liquid chromatography—mass spectrometry (LC—MS). The thermospray spectra of these compounds exhibited [M + H]⁺ and [M + Na]⁺ ions along with several fragment ions. The limits of detection for these antibiotics were estimated to be 50 to 100 ppb for LC with UV-PDA detection and 100–200 ppb for thermospray LC—MS detection.     

Patterns of clometacillin circulation in the body of laboratory animals]

Distribution of clometacillin in mice, rats and rabbits was studied in comparison with some other penicillins. It was found that clometacillin was superior to all other penicillins used for comparison with respect to the circulation time in the blood after intravenous, intramusclar or intrastomach administration. As for the capacity for penetrating into the tissues from the blood, clometacillin was not inferior to ampicillin and benzylpenicillin and was superior to propicillin, though it was bound by the blood serum proteins to a greater extent than the other penicillins used for comparison.     

A spectrophotometric assay of β-lactamase action on penicillins (Short Communication)

A new method for measuring the enzymic hydrolysis of the beta-lactam ring in penicillins is described. The change in extinction in the u.v. region is determined. The method is sensitive (50mum-benzylpenicillin can be used) and convenient.     

Early discoveries in the penicillin series.

It is now 50 years since the therapeutic potential of penicillin was first demonstrated. This first antibiotic and the series of compounds derived from it have been of immense importance in modern medicine. This article describes the early search for more potent penicillin derivatives, culminating with the discovery of an intermediate of penicillin biosynthesis, 6-beta-amino penicillanic acid (6-APA). A companion article in next month's TIBS will chart the subsequent exploitation of 6-APA and the preparation of a range of clinically important semi-synthetic penicillins.     

A practical design of an elution system for preparative electrophoresis in polyacrylamide gel.

The previously applied direct spectrophotometric assay of β-lactamase activity toward cephalosporins was found readily applicable to penicillins too. The differential uv absorption spectra of various penicillins and their corresponding penicilloic acids were determined. The appropriate experimental conditions were examined and the spectrophotometric assay seems adequate for the study of several substrates in a mixture. Also this method was found highly suitable for computerized analysis of the kinetic data for the determination of Michaelis constants of the various penicillins. The use of the integrated form of the rate equation for the evaluation of the best estimates of Michaelis constants was found advantageous.     

Determination of iodine-containing admixtures in semisynthetic penicillins

Dependence of the values defining the content of iodine sorbing admixtures in semisynthetic penicillins on pH, reaction time and drug aliquots was studied. On the basis of this study a general approach to development of procedures for determining iodine sorbing admixtures in semisynthetic penicillins was suggested. Procedures for determination of iodine sorbing admixtures in carbenicillin, carfecillin, ampicillin, oxacillin, azlocillin and ampiox were developed.     

Current problems of work hygiene in the production of semisynthetic penicillins

The hygienic pattern of the technological process for production of semisynthetic penicillins is described. It is shown that the chemical factor is the leading one of the environment in manufacture of semisynthetic penicillins unlike production of natural antibiotics. The characteristics of the chemical factor by the conditions of its creation, physicochemical properties and effect on man is presented. A set of hygienic recommendations for safe performance of the technological process for production of semisynthetic penicillins was developed and is now being introduced.     

Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.

Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.     

Specific Assay of Aminoglycosidic- or Polymyxin-Type Antibiotics Present in Human Sera in Combination with Cephalosporins

Monitoring of serum concentrations of aminoglycosidic or polymyxin antibiotics when administered concurrently with cephalosporins or penicillins requires a special assay technique. Selective enzymatic degradation of the beta-lactam antibiotic from the serum specimen allows subsequent assay of the antibiotic being monitored. This report gives details of a simple procedure for laboratory production of a crude enzyme capable of degrading cephalosporins or penicillins. An assay procedure for quantitating aminoglycosidic or polymyxin antibiotics after enzymatic degradation of a coexisting beta-lactam antibiotic is described.     

Advances in enzymatic transformation of penicillins to 6-aminopenicillanic acid (6-APA)

This article elaborates on the important recent developments in the enzymatic transformation of penicillins to 6-aminopenicillanic acid (6-APA), which is the basic raw material for the industrial production of semisynthetic penicillins such as amoxycillin and ampicillin. Particular emphasis is placed on the improvements in purification, stability, and immobilization of the enzymes, (i.e. penicillin acylases) used for these transformations.     

Radical‐scavenging activity of penicillin G,ampicillin, oxacillin,and dicloxacillin

The aim of this study was to characterize the antioxidant activity of penicillin G (PG), ampicillin (AMP), oxacillin (OX) and dicloxacillin (DOX) through their reactivity towards reactive oxygen species (superoxide anion radical, ; hydroxyl radical, HO^?; peroxyl radical, ROO^?; hydrogen peroxide, H₂O₂; DPPH^?) using various in vitro antioxidant assays with chemiluminescence (CL) and spectrophotometry as measurement techniques. In hydroxyl radical assays , PG, OX and AMP were found to inhibit the CL signal arising from the Fenton‐like reaction in a dose‐dependent manner with IC₅₀ = 0.480 ± 0.020 mM, IC₅₀ = 0.569 ± 0.021 mM, and IC₅₀ = 0.630 ± 0.019 mM, respectively. The highest reactivity of PG among the tested penicillins towards the HO radical was confirmed in the deoxyribose degradation assay. In the ABAP‐derived ROO radical assay, the radical‐scavenging ability of the test penicillins was in the following order: AMP > PG > DOX > OX. The number of reduced DPPH radicals by the drugs tested was <1 being the biggest for PG. The weak antioxidant capacity of the test penicillins was confirmed in the trolox antioxidant capacity assay (0.075 ± 0.004; 0.093 ± 0.006; 0.123 ± 0.005; 0.126 ± 0.004) for OX, AMP, DOX, PG, respectively. Use of luminol as a CL probe for estimation of penicillin reactivity towards H₂O₂ showed that only AMP was able to quench light emission; the remaining antibiotics demonstrated a strong enhancing effect. All the examined compounds showed a weak antioxidant potential when estimated using the ferric‐ferrozine assay. This study is the first to report the evaluation of test penicillins as antioxidants under the same reaction conditions.     

Formation of aminoxyl radicals in the reaction between penicillins and hydrogen peroxide.

Aminoxyl radicals are formed in high yield in the reaction between penicillins and hydrogen peroxide in water solutions in the pH range between 7 and 8. The nine-line EPR spectrum, 3 x 3 (1:2:1), indicated an interaction of the unpaired electron with one 14N nucleus (aN = 1.44 mT) and two equivalent hydrogen nuclei (aH = 2.00 mT). The reaction involves an oxidative cleavage of the beta-lactam ring of the penicillins with the formation of a cyclic aminoxyl radical, in which the thiazolidine ring carries the nitroxide group (= N-O.). It is suggested that the reaction with the formation of aminoxyl radicals can also take place in vivo in the deactivation of penicillins by metabolically formed hydrogen peroxide.     

Isolation and characterization of the acetyl-CoA synthetase from Penicillium chrysogenum. Involvement of this enzyme in the biosynthesis of penicillins.

Acetyl-CoA synthetase (ACS) of Penicillium chrysogenum was purified to homogeneity (745-fold) from fungal cultures grown in a chemically defined medium containing acetate as the main carbon source. The enzyme showed maximal rate of catalysis when incubated in 50 mM HCl-Tris buffer, pH 8.0, at 37 degrees C. Under these conditions, ACS showed hyperbolic behavior against acetate, CoA, and ATP; the Km values calculated for these substrates were 6.8, 0.18, and 17 mM, respectively. ACS recognized as substrates not only acetate but also several fatty acids ranging between C2 and C8 and some aromatic molecules (phenylacetic, 2-thiopheneacetic, and 3-thiopheneacetic acids). ATP can be replaced by ADP although, in this case, a lower activity was observed (37%). ACS in inhibited by some thiol reagents (5,5'-dithiobis(nitrobenzoic acid), N-ethylmaleimide, p-chloromercuribenzoate) and divalent cations (Zn2+, Cu2+, and Hg2+), whereas it was stimulated when the reaction mixtures contained 1 mM dithiothreitol, reduced glutathione, or 2-mercaptoethanol. The calculated molecular mass of ACS was 139 +/- 1 kDa, and the native enzyme is composed of two apparent identical subunits (70 kDa) in an alpha 2 oligomeric structure. ACS activity was regulated "in vivo" by carbon catabolite inactivation when glucose was taken up by cells in which the enzyme had been previously induced. This enzyme can be coupled "in vitro" to acyl-CoA:6-aminopenicillanic acid acyltransferase from P. chrysogenum, thus allowing the reconstitution of the functional enzymatic system which catalyzes the two latter reactions responsible for the biosynthesis of different penicillins. The ACS from Aspergillus nidulans can also be coupled to 6-aminopenicillanic acid acyltransferase to synthesize penicillins. These results strongly indicate that this enzyme can catalyze the activation (to their CoA thioesters) of some of the side-chain precursors required in these two fungi for the production of several penicillins. All these data are reported here for the first time.     

Distribution of sulfanilamides and penicillins in the blood and organs of rats after their combined administration

The use of benzylpenicillin and ampicillin in combination with sulfalen or sulfadimethoxine increased the levels of the penicillins and sulfalen in some organs and tissues of rats. This was accompanied by a rise in the concentration gradients of the drugs. It is concluded that the combined use of the penicillins and sulfanilamides determines their increased penetration from the blood into other organs and tissues of the host.     

Enzyme-Linked Immunosorbent Assay of Ampicillin in Milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10–1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. Limit of detection for ampicillin in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).     

Industrial production of beta-lactam antibiotics

The industrial production of beta-lactam antibiotics by fermentation over the past 50 years is one of the outstanding examples of biotechnology. Today, the beta-lactam antibiotics, particularly penicillins and cephalosporins, represent the world's major biotechnology products with worldwide dosage form sales of approximately 15 billion US dollars or approximately 65% of the total world market for antibiotics. Over the past five decades, major improvements in the productivity of the producer organisms, Penicillium chrysogenum and Acremonium chrysogenum (syn. Cephalosporium acremonium) and improved fermentation technology have culminated in enhanced productivity and substantial cost reduction. Major fermentation producers are now estimated to record harvest titers of 40-50 g/l for penicillin and 20-25 g/l for cephalosporin C. Recovery yields for penicillin G or penicillin V are now >90%. Chemical and enzymatic hydrolysis process technology for 6-aminopenicillanic acid or 7-aminocephalosporanic acid is also highly efficient (approximately 80-90%) with new enzyme technology leading to major cost reductions over the past decade. Europe remains the dominant manufacturing area for both penicillins and cephalosporins. However, due to ever increasing labor, energy and raw material costs, more bulk manufacturing is moving to the Far East, with China, Korea and India becoming major production countries with dosage form filling becoming more dominant in Puerto Rico and in Ireland.