Kinetic studies of a beta-lactamase by a computerized microacidimetric method.

On-line computerized treatment of enzyme kinetic data allows the precise measurement of Michaelis--Menten constants (Km and V) from a single progress curve. This method has been used to determine the kinetic constants of a beta-lactamase extracted from an Escherichia coli strain. In the profile of enzymatic activity there obtained, Km and V are a function of the pH. From these results some information is derived about the mechanism of the enzyme--substrate binding.

A new approach for using cofactor dependent enzymes: example of alcohol dehydrogenase.

The use of enzymes requiring a cofactor as substrate in organic synthesis is still a problem since the cofactors are expensive. This study deals with a new approach consisting of using fragments of NAD+. Three fragments of NAD(H) are examined. The activities of NMN+ and NMNH are greatly improved by the addition of adenosine in ethanol oxidation and in cyclohexanone reduction, respectively. Nicotinamide mononucleoside is not active in the ethanol oxidation but the addition of AMP promotes this reaction.

[Immobilization of coenzyme A on a hydrosoluble polymer]. / Immobilisation du coenzyme A sur polymère hydrosoluble.

Our purpose being to work out a "continuous flow" enzymatic reactor of acetylation with a cofactor, we present here some results about the immobilization of coenzyme A on T40 dextran. Two methods of fixation are compared with regard to the capacity and the biological activity of the polymers thus obtained.

[Purification of beta-lactamases by affinity chromatography]. / Purification des beta-lactamases par chromatographie d'affinité

Affinity columns able to purifie beta-lactamases have been prepared either by linking covalently reversible inhibitors or substrates to agarose beds. The enzyme is eluted with a gradient of sodium chloride or released with the substrate. This method is a pertinent one for the purification of these enzymes and for the study of bacteria harbouring more than one beta-lactamase.

[Partial synthesis of aminoglycosidic antibiotics. I. An enzymatic reactor model using cofactors]. / Hémisynthèse d'antibiotiques aminoglycosidiques: I. - Mise au point d'un réacteur enzymatique à cofacteur.

In order to produce specifically N-monoalkylated derivatives of aminoglycoside antibiotics of potential therapeutic values, we have developed an enzymatic reactor. This system uses the aminoglycoside acetyltransferase as catalyst and acetylcoenzyme A as acetyl donor. The immobilization of one aminoglycoside acetyltransferase on different resins has been studied. The coreticulation of this enzyme on DEAE cellulose in the presence of glutaraldehyde gives rise to an enzymatic resin of high efficiency. On the other hand, we have also studied the acetylation of coenzyme A in a simple manner. Acetylation occurs in a quantitative yield when the reaction is performed in the presence of polyvinyl-4 pyridine/divinylbenzene 2 per cent. These conclusions enabled to develop two types of acetylating reactors which give rise without purification to 3-acetyl gentamicin.

[Identification of the beta lactamase R-TEM of Pseudomonas aeruginosa]. / Identification de la beta lactamase R-TEM chez Pseudomonas aeruginosa

This paper is dealing with the enzymatic problem raised by two strains of Ps. aeruginosa resistant to classical beta lactam antibiotics including carbenicillin. These two strains hydrolyse all these antibiotics. In both cases, we have shown the simultaneous biosynthesis of two enzymes: an inducible and chromosome cephalosporinase frequently found in this germ, and a constitutive beta lactamase, with a penicillinase activity which has been identified with the extrachromosomic beta lactamase R-TEM. These two enzymes have been separated by affinity chromatography, characterized by their kinetic constants given by computerized microacidimetry, and their isoelectric points which are respectively 9.2 for the cephalosporinase and 5.40 for the penicillinase R-TEM. Isoelectric focussing also shows the separation of these two enzymes.

(1-Amino-2-propenyl) phosphonic acid, an inhibitor of alanine racemase and D-alanine:D-alanine ligase.

DL-(1-Amino-2-propenyl)phosphonic acid was synthesized through the sequential oxidation, sulfoxide elimination, and deprotection of diphenyl [1-[(benzyloxycarbonyl)amino]-3-(phenylthio)propyl] phosphonate. This analogue of vinylglycine is a strong inhibitor of the alanine racemases from Pseudomonas aeruginosa and Streptococcus faecalis and of the D-Ala:D-Ala ligase from this latter species. This molecule is ineffective against the whole bacterial cells. Unlike vinylglycine, this unsaturated phosphonate does not inhibit the following mammalian enzymes: aspartate aminotransferase, alanine aminotransferase, D-amino acid oxidase, which indicates its specificity. Thus, its incorporation in a peptide structure could induce interesting antimicrobial properties.

(beta-Chloro-alpha-aminoethyl)phosphonic acids as inhibitors of alanine racemase and D-alanine:D-alanine ligase.

The (beta-chloro-, (beta, beta-dichloro-, and (beta, beta, beta-trichloro-alpha-aminoethyl)phosphonic acids have been synthesized and their inhibitory properties on the alanine racemases [EC 5.1.1.1] and the D-Ala:D-Ala ligases [EC 6.3.2.4] from Pseudomonas aeruginosa and Streptococcus faecalis have been evaluated. The monochloro and the dichloro derivatives of Ala-P exhibit a strong inhibition on the racemases of the two species tested but do not behave as suicide substrates. Only the D-Ala:D-Ala ligase of S. faecalis is inhibited by these compounds. The poor antibacterial activity observed with beta-chloro- and beta, beta-dichloro-Ala-P might be enhanced by the peptide-transport strategy.

The lysine pathway as a target for a new genera of synthetic antibacterial antibiotics?

Unsaturated analogues of diaminopimelic acid have been synthesized. The amino acids were designed so that they would be reversible or irreversible inhibitors of both of the two last enzymes of the lysine pathway. The compounds were tested with meso-diaminopimelate decarboxylase. trans-3,4-Didehydrodiaminopimelic acid (2) was found to be the most potent inhibitor. The antibacterial activities did not correlate with enzyme inhibiting activities. 4-Methylenediaminopimelic acid 4 showed strong antibacterial properties. It is suggested that L,L-diaminopimelate epimerase could be the target enzyme.

Purification and characterization of the main pepsinogen from the shark, Centroscymnus coelolepis.

The main pepsinogen from the mucosa of the shark, Centroscymnus coelolepis, has been purified and characterized. This zymogen, the most abundant protein in terms of quantity and activity (yield 72%), is a homogeneous monomer of molecular weight 42+/-0.7 kDa, as determined by electrophoresis. The aspartyl proteinase nature of this enzyme was confirmed by the considerable inhibition by pepstatin. Its specificity as to the oxidized B-chain of bovine insulin was determined using electrospray ionization mass spectrometry (ESI-MS) coupled with reversed phase high pressure liquid chromatography (RP-HPLC). The 15-16 Leu-Tyr bond was rapidly cleaved in this substrate, followed by the 24-25 Phe-Phe, 25-26 Phe-Tyr, and 11-12 Leu-Val bonds.

Pristinamycin accumulation by Staphylococcus aureus.

Pristinamycins IA and IIA (PIA and PIIA) accumulation by Staphylococcus aureus has been studied with two hydrogenated analogs, (H2)PIA and (H2)PIIA. Rapid accumulation of both antibiotics at 37 degrees C is observed and internal concentrations can reach up to 58-fold the external concentration; this accumulation cannot be reduced by either metabolic inhibitors or tetracycline. The synergistic activity of pristinamycins IA and IIA is not observed at the bacterial accumulation level. We propose that pristinamycins enter into bacteria by a passive diffusion process and that the internal concentration is maintained by binding of the antibiotic to the bacterial ribosomes.

Have deoxystreptamine aminoglycoside antibiotics the same binding site on bacterial ribosomes?

(3H) Tobramycin was used as a probe to determine the relationship between the structure of aminoglycoside antibiotics and their ability to remove this drug from its higher affinity binding site on the ribosome. The dissacharide moieties (neamine, tobramine, gentamine) appeared to have a common binding site, whereas the kanosamine, garosamine and ribose moieties determined the specificity of this binding. Amikacin and butikacin behaved in an anomalous manner in spite of their close structural relationship to tobramycin.

Effects of cations, polyamines and other aminoglycosides on gentamicin C2. Binding to ribosomes from sensitive and resistant Escherichia coli strains.

Gentamicin C2 interacts cooperatively with ribosomes from a sensitive Escherichia coli strain in a multiphasic way with several classes of sites. It is shown that this binding is highly-dependent on Mg++ and natural endogenous polyamine concentrations. The differences observed between ribosomes from sensitive and resistant strains may be explained by the absence of specific cooperative gentamicin interactions with resistant ribosomes. The effects of other aminoglycoside antibiotics are discussed in terms of structure-activity relationships.

Studies on pristinamycin synergism in Staphylococcus aureus.

Binding experiments were performed with both components of the pristinamycin complex (pristinamycin IA (PIA) and pristinamycin IIA (PIIA] using ribosomes from sensitive and resistant Staphylococcus aureus. Fluorescence polarization was used to measure PIA binding. The results obtained show a direct correlation between inhibition, synergy and the enhancement of the affinity of PIA for its receptor in the presence of PIIA. The uptake of PIA by intact cells seems to be directly correlated with affinity between PIA and ribosomes, a phenomenon which is probably shared with the macrolide antibiotics.

Irreversible binding of pristinamycin IIA (streptogramin A) to ribosomes explains its "lasting damage" effect.

In vitro and in vivo studies are presented to test the hypothesis that the synergistic action of the pristinamycins is not due to a catalytic effect of pristinamycin IIA (PIIA) on the bacterial ribosome. We demonstrate that there is a proportionality between the quantity of PIIA bound on the ribosome and pristinamycin IA (PIA) retained by it. Moreover in vitro and in vivo experiments correlated to biological effects (growth and protein synthesis) demonstrate that pristinamycin IIA is tightly bound on 70S ribosome, which satisfactory explains the so called "lasting damage effect".

Synthesis of pseudotrisaccharides related to ribostamycin.

The three protected sisamine derivatives 2i, 2j and 3, with a free 5-hydroxyl group, have been synthesized. Glycosylation at the 5 position with various pentofuranose derivatives yielded after deprotection of the 6a approximately i ribostamycin related aminoglycoside. These pseudotrisaccharides showed only low antibacterial activities with respect to the parent compounds.

Binding of tobramycin to Escherichia coli ribosomes: characteristics and equilibrium of the reaction.

A sample of [3H] tobramycin (5,000 Ci/Mole) has been synthetized and incubated with the bacterial ribosome and its subunits. The results obtained show that this antibiotic has two types of binding sites. The primary one is probably responsible for the inhibition of protein synthesis whereas the secondary one is probably related to the misreading and reading through of the messenger RNA.

Mechanism of action of a 16-membered macrolide. Characteristics of dihydrorosaramicin binding to Escherichia coli ribosome and the effects of some competitors.

The macrolide [3H]dihydrorosaramicin binds specifically to 50S and 70S bacterial ribosomal particles. We have studied the influence of salts, pH and additives on the interaction and found that the optimum requirement for salts was 10 mM trs-HCl (pH 7.6), 6 mM MgCl2, 60 mM NH4Cl, and that beta-mercaptoethanol which reacts on rosaramicin and its dihydro derivative cannot be used. The parameters of the binding were not dependent on the technique used, i.e. equilibrium dialysis, ethanol precipitation or two-phase partitioning. In our search for effectors of this binding, we have found that it is inhibited by other macrolides, little effected by tobramycin and chloramphenicol and enhanced by puromycin.

Synthesis of sisamine and of pseudodisaccharide analogues.

Lividamine and paromamine were converted into two key intermediate ethylenic aldehydes 10a and 10b. Reductive amination of the two aldehydes yielded the protected sisamine 11a and the three analogs 11b, 12a and 12b. These four derivatives were deprotected to yield the four pseudodisaccharides 1a, 1b, 2a and 2b which were less active in vitro than neamine against Escherichia coli ATCC 9637 and Staphylococcus aureus 209P.

Effects of N-alkylation and n-acylation on tobramycin activity.

The activities of tobramycin derivatives acetylated and ethylated on the 6'-N,2'-N and 3-N positions were examined. The MICs of these derivatives against tobramycin sensitive strains indicated that 2'-N-ethylated and 6'-N-ethylated derivatives have a fairly good activity, and confirmed that the 3-N position is the most important one for antibiotic activity since 3-N derivatives were less active. The MICs of these derivatives against tobramycin resistant strains, and their inactivation by tobramycin modifying enzymes were examined. These results showed that 2'-N or 6'-N ethylation protects the drug against inactivation by AAC(2') or AAC(6'), respectively, and 2'-N-ethyltobramycin and 6'-N-ethyltobramycin were active against strains containing these modifying enzymes. On the other hand, 3-N ethylation protects the drug against inactivation by AAC(3) but 3-N-ethyl tobramycin does not inhibit strains containing this enzyme.