The Mechanisms of Catalysis by Metallo β-Lactamases

Class B β-lactamases or metallo-β-lactamases (MBLs) require zinc ions to catalyse the hydrolysis of β-lactam antibiotics such as penicillins, cephalosporins, carbapenems, and cephamycins. There are no clinically useful inhibitors against MBLs which are responsible for the resistance of some bacteria to antibiotics. There are two metal-ion binding sites that have different zinc ligands but the exact roles of the metal-ion remain controversial, and distinguishing between their relative importance is complex. The metal-ion can act as a Lewis acid by co-ordination to the β-lactam carbonyl oxygen to facilitate nucleophilic attack and stabilise the negative charge developed on this oxygen in the tetrahedral intermediate anion. The metal-ion also lowers the pKa of the directly co-ordinated water molecule so that the metal-bound hydroxide ion is a better nucleophile than water and is used to attack the β-lactam carbonyl carbon. An intrinsic property of binuclear metallo hydrolytic enzymes that depend on a metal-bound water both as the attacking nucleophile and as a ligand for the second metal-ion is that this water molecule, which is consumed during hydrolysis of the substrate, has to be replaced to maintain the catalytic cycle. With MBL this is reflected in some unusual kinetic profiles

Reactivity and inhibition of metallo-β-lactamases

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Enzyme deactivation due to metal-ion dissociation during turnover of the cobalt β-lactamase catalysed hydrolysis of β-lactams

Metallo-β-lactamases are native zinc enzymes that catalyze the hydrolysis of -lactam antibiotics but are also able to function with cobalt (II) and require one or two metal ions for catalytic activity. The kinetics of the hydrolysis of benzylpenicillin catalyzed by cobalt substituted -lactamase from Bacillus cereus (BcII) are biphasic. The dependence of enzyme activity on pH and metal-ion concentration indicates that only the di-cobalt enzyme is catalytically active. A mono-cobalt enzyme species is formed during the catalytic cycle, which is virtually inactive and requires the association of another cobalt ion for turnover. Two intermediates with different metal to enzyme stoichiometries are formed on a branched reaction pathway. The di-cobalt enzyme intermediate is responsible for the direct catalytic route, which is pH-independent between 5.5 and 9.5 but is also able to slowly lose one bound cobalt ion via the branching route to give the mono-cobalt inactive enzyme intermediate. This inactivation pathway of metal-ion dissociation occurs by both an acid catalyzed and a pH-independent reaction, which is dependent on the presence of an enzyme residue of pKa = 8.9 ± 0.1 in its protonated form and shows a large kinetic solvent isotope effect (H2O/D2O) of 5.2 ± 0.5, indicative of a rate-limiting proton transfer. The pseudo first-order rate constant to regenerate the di-cobalt -lactamase from the mono-cobalt enzyme intermediate has a first-order dependence on cobalt-ion concentration in the pH range 5.5-9.5. The second-order rate constant for metal-ion association is dependent on two groups of pKa 6.32 ± 0.1 and 7.47 ± 0.1 being in their deprotonated basic forms and one group of pKa 9.48 ± 0.1 being in its protonated for

Loss of enzyme activity during turnover of the Bacillus cereus β-lactamase catalysed hydrolysis of β-lactams due to loss of zinc ion

Metallo-β-lactamases are zinc-ion-dependent and are known to exist either as mononuclear or as dinuclear enzymes. The kinetics and mechanism of hydrolysis of the native zinc Bacillus cereus metallo-β-lactamase (BcII) have been investigated under pre-steady-state conditions at different pHs and zinc-ion concentrations. Biphasic kinetics are observed for the hydrolysis of cefuroxime and benzylpenicillin with submicromolar concentrations of enzyme and zinc. The initial burst of product formation far exceeds the concentration of enzyme and the subsequent slower rate of hydrolysis is attributed to a branched kinetic pathway. The pH and metal-ion dependence of the microscopic rate constants of this branching were determined, from which it is concluded that two enzyme species with different metal-to-enzyme stoichiometries are formed during catalytic turnover. The dizinc enzyme is responsible for the fast route but during the catalytic cycle it slowly loses the less tightly bound zinc ion via the branching route to give an inactive monozinc enzyme; the latter is only catalytic following the uptake of a second zinc ion. The rate constant for product formation from the dinuclear enzyme and the branching rate constant show a sigmoidal dependence on pH indicative of important ionizing groups with pK as of 9.0 ± 0.1 and 8.2 ± 0.1, respectively. The rate constant for the regeneration of enzyme activity depends on zinc-ion concentration. This unusual behaviour is attributed to an intrinsic property of metallo hydrolytic enzymes that depend on a metal bound water both as a ligand for the second metal ion and as the nucleophile which is consumed during hydrolysis of the substrate and so has to be replaced to maintain the catalytic cycle

The variation of catalytic efficiency of bacillus cereus metallo- β-lactamase with different active site metal ions

The kinetics and mechanism of hydrolysis of the native zinc and metal substituted Bacillus cereus (BcII) metallo--lactamase have been investigated. The pH and metal ion dependence of kcat and kcat/Km, determined under steady-state conditions, for the cobalt substituted BcII catalyzed hydrolysis of cefoxitin, cephaloridine, and cephalexin indicate that an enzyme residue of apparent pKa 6.3 ± 0.1 is required in its deprotonated form for metal ion binding and catalysis. The kcat/Km for cefoxitin and cephalexin with cadmium substituted BcII is dependent on two ionizing groups on the enzyme: one of pKa1 = 8.7 ± 0.1 required in its deprotonated form and the other of pKa2 = 9.3 ± 0.1 required in its protonated form for activity. The pH dependence of the competitive inhibition constant, Ki, for CdBcII with L-captopril indicates that pKa1 = 8.7 ± 0.1 corresponds to the cadmium-bound water. For the manganese substituted BcII, the pH dependence of kcat/Km for benzylpenicillin, cephalexin, and cefoxitin similarly indicated the importance of two catalytic groups: one of pKa1 = 8.5 ± 0.1 which needs to be deprotonated and the other of pKa2 = 9.4 ± 0.1 which needs to be protonated for catalysis; the pKa1 was assigned to the manganese-bound water. The rate was metal ion concentration dependent at the highest manganese concentrations used (10-3 M). The metal substituted species have similar or higher catalytic activities compared with the zinc enzyme, albeit at pHs above 7. Interestingly, with cefoxitin, a very poor substrate for ZnBcII, both kcat and kcat/Km increase with increasing pKa of the metal-bound water, in the order Zn < Co < Mn < Cd. A higher pKa for the metal-bound water for cadmium and manganese BCII leads to more reactive enzymes than the native zinc BcII, suggesting that the role of the metal ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediat

Conception, synthèse et évaluation biologique de nouvelles classes de ligands sérotoninergiques 5-HT7

Parmi tous les neurotransmetteurs identifiés à ce jour, la sérotonine (5-hydroxytryptamine, 5-HT) est impliquée dans le système le plus complexe de récepteurs. Parmi eux, les récepteurs 5-HT7 qui sont les derniers découverts (1993) semblent avoir des implications multiples tant au niveau central que périphérique. Le potentiel thérapeutique représenté par la découverte de ligands 5-HT7 sélectifs vis-à-vis d autres RCPGs a motivé notre projet de recherche. Nos études sont orientées vers la conception de trois classes distinctes de ligands. Une première famille à été conçue sur une charpente benzimidazolone. Diverses pharmacomodulations ont permis un changement du profil d activité de 5-HT1A vers 5-HT7. Une deuxième famille de composés à structure furo- ou pyrano[2,3-b] pyridinique constitue des analogues azotés d un des plus intéressants agonistes sélectifs 5-HT7 actuels. La synthèse de ces dérivés a été conduite via la mise en oeuvre d une étape clé de cycloaddition intramoléculaire de Diels-Alder à partir de 1,2,4-triazines judicieusement fonctionnalisées en 3 par une chaine aminoalkynol. Cette méthodologie nous a permis de faire varier les substituants alkyle de l amine, la nature et la position du motif aryle sur le noyau pyridinique, ainsi que la taille du cycle non-aromatique. La synthèse d une dernière famille de dérivés bisaryliques a enrichi les études de relation structure-activité, connues dans la littérature, associées à ce type de ligands 5-HT7. La variation du cycle aromatique central (phényle, 1,3-diazine et 1,2,4- triazine) a révélé d importantes conséquences sur l affinité des molécules.Among all the neurotransmitters identified up-to-date, serotonin (5-Hydroxytryptamine, 5-HT) is mediated by the most complex system of receptors. The 5-HT7 receptors are the latest discovered (1993) and have many implications both in the central nervous system and in peripheral tissues. The therapeutic potential of new 5- HT7 ligands, selective over the other GPCRs, motivated our research project. Our studies were focused on the design of three different classes of 5-HT7 ligands. The first class was built on a benzimidazolone scaffold. Various modulations afforded a shift of the affinity profile from the 5-HT1ARs to the 5-HT7Rs. A second class of 3-aminofuro- or pyrano[2,3-b]pyridines are in fact the heteroanalogues of one of the most interesting current 5-HT7 selective agonists: the 3-aminochromans. Their synthesis involved an intramolecular Diels-Alder cycloaddition key step starting from a 1,2,4-triazine judiciously substituted in 3 with a convenient aminoalkynol. The developed methodology afforded the variation of the substituents on the amine moiety, of the aromatic substituent and its position on the pyridinic core and of the non-aromatic ring size. A last class of bisarylic derivatives further explored the SAR tendencies of this type of 5-HT7 ligands by modulating the main aromatic scaffold in the benzene, pyrimidine and 1,2,4-triazine series.ORLEANS-SCD-Bib. electronique (452349901) / SudocSudocFranceF

Thermodynamics of copper and zinc distribution in the cyanobacterium Synechocystis PCC 6803

Copper is supplied to plastocyanin for photosynthesis and cytochrome c oxidase for respiration in the thylakoids of Synechocystis PCC 6803 by the membrane-bound P-type ATPases CtaA and PacS, and the metallochaperone Atx1. We have determined the Cu(I) affinities of all of the soluble proteins and domains in this pathway. The Cu(I) affinities of the trafficking proteins range from 5 × 1016 to 5 × 1017 M-1 at pH 7.0, consistent with values for homologues. Unusually, Atx1 binds Cu(I) significantly tighter than the metal-binding domains (MBDs) of CtaA and PacS (CtaAN and PacSN), and equilibrium copper exchange constants of approximately 0.2 are obtained for transfer to the MBDs. Dimerization of Atx1 increases the affinity for Cu(I), but the loop 5 His61 residue has little influence. The MBD of the zinc exporter ZiaA (ZiaAN) exhibits an almost identical Cu(I) affinity, and Cu(I) exchange with Atx1, as CtaAN and PacSN, and the relative stabilities of the complexes must enable the metallochaperone to distinguish between the MBDs. The binding of potentially competing zinc to the trafficking proteins has been studied. ZiaAN has the highest Zn(II) affinity and thermodynamics could be important for zinc removal from the cell. Plastocyanin has a Cu(I) affinity of 2.6 × 1017 M-1, 15-fold tighter than that of the CuA site of cytochrome c oxidase, highlighting the need for specific mechanisms to ensure copper delivery to both of these targets. The narrow range of Cu(I) affinities for the cytoplasmic copper proteins in Synechocystis will facilitate relocation when copper is limiting