Inactivation of penicillin acylase from Kluyvera citrophila by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline: a case of time-dependent non-covalent enzyme inhibition

Abstract

Penicillin acylase (PA) from Kluyvera citrophila was inhibited by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a specific carboxy-group-reactive reagent. Enzyme activity progressively decreased to a residual value depending on EEDQ concentration. Neither enzymic nor non-enzymic decomposition of EEDQ is concomitant with PA inactivation. Moreover, enzyme re-activation is achieved by chromatographic removal of EEDQ, pH increase or displacement of the reagent with penicillin G. It was then concluded that PA inactivation is due to an equilibrium reaction. The kinetics of enzyme inactivation was analysed by fitting data to theoretical equations derived in accordance with this mechanism. Corrections for re-activation during the enzyme assay were a necessary introduction. The pH-dependence of the rate constant for EEDQ hydrolysis either alone or in the presence of enzyme was studied by u.v. spectroscopy. It turned out to be coincident with the pH-dependence of the forward and reverse rate constants for the inactivation process. It is suggested that previous protonation of the EEDQ molecule is required for these reactions to occur. The thermodynamic values associated with the overall reaction showed little change. Finally it is proposed that the inactivation of PA by EEDQ proceeds through a two-step reaction. The initial and rapid reversible binding is followed by a slow, time-dependent, non-covalent, reversible inactivating step. The expected behaviour in the case of enzyme modification by covalent activation of carboxy residues is also reviewed.

Behavioral sensitization to cocaine, but not cocaine-conditioned behavior, is associated with increased dopamine occupation of its receptors in the nucleus accumbens.

“…Only those sites not already bound with a ligand are apparently vulnerable to EEDQ-induced inactivation. This apparent competition is consistent with a two-step mechanism of action for EEDQ, with the first step being a rapid and competitive binding to a recognition site, and the second step being a very slow reaction, and hence virtually irreversible, as has been described for the inactivation of an enzyme by EEDQ (Martin, Mancheño, & Arche, 1993). Protection from EEDQ-induced reduction in the density of dopamine receptors therefore provides an indication of in vivo occupation of both D 1 -like and D 2 -like receptor subtypes by endogenous and/or exogenous ligands (Burger & Martin-Iverson, 1993, 1994; Martin-Iverson & Burger, 1995).…”

Antimicrobial Coatings with Dual Cationic and N-Halamine Character: Characterization and Biocidal Efficacy

“…The 2 × 2 cm coupons were UV-ozone-irradiated from one side with a Jelight Co. model 42 UVO Cleaner (Irvine, CA, USA) for 15 min to create carboxylic acid groups on their surfaces , (this step will be referred as UV-O 3 ). To create surface anhydride groups (and make the PP surface reactive toward the primary amines of PEI through nucleophilic attack), UV-O 3 coupons were shaken for 2 h at room temperature in a 0.1 mM solution of EEDQ in 50 mM MES buffer (pH 5.5) . Before mixing with the MES buffer, the necessary amount of EEDQ was dissolved in a volume of methanol equal to <1% of the final volume of the mix.…”

“…To create surface anhydride groups (and make the PP surface reactive toward the primary amines of PEI through nucleophilic attack), UV-O 3 coupons were shaken for 2 h at room temperature in a 0.1 mM solution of EEDQ in 50 mM MES buffer (pH 5.5). 30 Before mixing with the MES buffer, the necessary amount of EEDQ was dissolved in a volume of methanol equal to <1% of the final volume of the mix. This step will be referred to as EEDQ.…”

Coupling of Site‐Directed Mutagenesis and Immobilization for the Rational Design of More Efficient Biocatalysts: The Case of Immobilized 3G3K PGA from E.coli

“…[1,17] However, in spite of the advantages of this approach, the immobilization should not negatively interfere with the interaction of the substrates with the active centre and the conformational changes the enzyme undergoes therein. [18][19][20] Consequently, the influence of any structural change (i.e., a distortion of the amino acid residues involved in catalysis and/or in substrate binding) induced by immobilization cannot be underestimated. In fact, it has been reported that immobilization influences the catalytic properties of PGA, [21] depending on the type of matrix, the binding chemistry and the microenvironment created around the active site.…”