Serine lactamase

The following protocol is described for the selection of phage-displayed serine / -lactamase with a biotinylated penam-sulfone [5] suicide substrate. For other activities, the concentrations of substrate and suicide substrate and the times of reaction should probably be adjusted. 

This strategy has also been applied for the selection of active //-lactamases from a library of mutants also containing penicillin-binding proteins. For this purpose, the protocol had to be modified to circumvent a difficulty of selections with suicide substrates in mechanisms involving a covalent intermediate. If inhibition arises from a covalent intermediate (Y in Scheme 5.2, an acyl-enzyme in the case of serine //-lactamases), enzymes whose rate of release of this intermediate (hydrolysis of the acyl-enzyme) is slow will be efficiently selected as the efficiency of inhibition depends on the ratio of rate constants k4/k3 (Scheme 5.2). To prevent the selection of enzymes with inadequate turnover, a counter-selection step was included in the protocol the library of mutants was incubated with substrate in order to block them as covalent intermediates before adding the biotinylated inhibitor. The library could be enriched from 6 ppm to 25 % active //-lactamases in four rounds of selection [62]. 

An example for proteases are the (3-lactamases that hydrolyse a peptide bond in the essential (3-lactam ring of penicillins, cephalosporins, carbapenems and monobac-tams and, thereby, iireversibly inactivate the diug. 13-lactamases share this mechanism with the penicillin binding proteins (PBPs), which are essential enzymes catalyzing the biosynthesis of the bacterial cell wall. In contrast to the PBPs which irreversibly bind (3-lactams to the active site serine, the analogous complex of the diug with (3-lactamases is rapidly hydrolyzed regenerating the enzyme for inactivation of additional (3-lactam molecules. 

According to their genetic relationship and their biochemical mechanism of action (3-lactamases are divided into enzymes of the serine-protease type containing an active-site serine (molecular class A, C, and D enzymes) and those of the metallo-protease type (molecular class B enzymes), which contain a complex bound zinc ion. 

Antibiotic Resistance. Figure 1 According to Bush, Jacoby and Medeiros [2] four molecular classes of (3-lactamases can be discriminated based upon biochemical and molecular features. Classes 1, 2, and 4 included serine-proteases, while metallo enzymes are included in class 3. The substrate spectrum varies between different subclasses and the corresponding genes can be part of an R-plasmid leading to a wider distribution or are encoded chromosomally in cells of specific species. 

P-Lactamases are enzymes that hydrolyze the P-lactam ring of P-lactamantibiotics (penicillins, cephalosporins, monobactams and carbapenems). They are the most common cause of P-lactam resistance. Most enzymes use a serine residue in the active site that attacks the P-lactam-amid carbonyl group. The covalently formed acylester is then hydrolyzed to reactivate the P-lacta-mase and liberates the inactivated antibiotic. Metallo P-lactamases use Zn(II) bound water for hydrolysis of the P-lactam bond. P-Lactamases constitute a heterogeneous group of enzymes with differences in molecular structures, in substrate preferences and in the genetic localizations of the encoding gene (Table 1). 

Class A Serine p-lactamases SHV-1 penicillinase in K. pneumoniae, and Koxy with activity against certain third generation cephalosporins in K. oxytoca BlaZ staphylococcal penicillinase TEM, SHV, VEB, PER and CTX-M penicillinases and ESBLs (P-lactamases with activity against third generation cephalosporins and aztreo-nam) KPC, IMI/NMC and SME carbapenemases... 

Class C Serine p-lactamases AmpC enzymes of coti, Shigella spp., Enterobacterspp., C. freundii, M. morganii, Providencia spp. and Serratia spp. cephalos-porinases with wide spectrum of activity CMY, LAT, BIL, MOX, ACC, FOX and DHA types. All genes are ampC genes that have been mobilized by transfer to plasmid DNA. 

Class D Serine p-lactamases OXA enzymes (oxacillinases) of Acinetobac-ter spp. and some Aeromonas spp. Some OXA enzymes are carbapenemases Most OXA types are chromosomal... 

QUINONE METHIDES AND AZA-QUINONE METHIDES AS LATENT ALKYLATING SPECIES IN THE DESIGN OF MECHANISM-BASED INHIBITORS OF SERINE PROTEASES AND p-LACTAMASES... 

The starting point for much of the work described in this article is the idea that quinone methides (QMs) are the electrophilic species that are generated from ortho-hydro-xybenzyl halides during the relatively selective modification of tryptophan residues in proteins. Therefore, a series of suicide substrates (a subtype of mechanism-based inhibitors) that produce quinone or quinonimine methides (QIMs) have been designed to inhibit enzymes. The concept of mechanism-based inhibitors was very appealing and has been widely applied. The present review will be focused on the inhibition of mammalian serine proteases and bacterial serine (3-lactamases by suicide inhibitors. These very different classes of enzymes have however an analogous step in their catalytic mechanism, the formation of an acyl-enzyme intermediate. Several studies have examined the possible use of quinone or quinonimine methides as the latent... 

The antibiotic activity of certain (3-lactams depends largely on their interaction with two different groups of bacterial enzymes. (3-Lactams, like the penicillins and cephalosporins, inhibit the DD-peptidases/transpeptidases that are responsible for the final step of bacterial cell wall biosynthesis.63 Unfortunately, they are themselves destroyed by the [3-lactamases,64 which thereby provide much of the resistance to these antibiotics. Class A, C, and D [3-lactamases and DD-peptidases all have a conserved serine residue in the active site whose hydroxyl group is the primary nucleophile that attacks the substrate carbonyl. Catalysis in both cases involves a double-displacement reaction with the transient formation of an acyl-enzyme intermediate. The major distinction between [3-lactamases and their evolutionary parents the DD-peptidase residues is the lifetime of the acyl-enzyme it is short in (3-lactamases and long in the DD-peptidases.65-67... 

The functionalized phenaceturates 16 (Fig. 11.10) are substrates of class A and C [3-lactamases, especially the class C enzymes, as observed with the parent unfunctionalized phenaceturates 15. They are also modest inhibitors of these enzymes and the serine DD-peptidase of Streptomyces R61. The inhibition of class C [3-lactamases is turnover dependent, as expected for a mechanism-based inhibitor. Inhibition is not very dependent on the nature of the leaving group, suggesting that the QM is generated in solution after the product phenol has been released from the active site. It therefore... 

Pratt, R. F. Functional evolution of the serine P-lactamase active site. J. Chem. Soc. Perkin Trans. 2 2002, 851-861. 

Govardhan, C. P. Pratt, R. F. Kinetics and mechanism of the serine P-lactamase catalyzed hydrolysis of depsipeptides. Biochemistry 1987, 26, 3385-3395. 

The mechanism of serine (3-lactamases is similar to that of a general serine hydrolase. Figure 8.14 illustrates the reaction of a serine (3-lac(amasc with another type of (3-lactam antibiotic, a cephalosporin. The active-site serine functions as an attacking nucleophile, forming a covalent bond between the serine side chain oxygen... 

Figure 8.14 Reaction mechanism for hydrolysis of a cephalosporin by a serine 3-lactamase.

Figure 8.16 Reaction of a serine (3-lactamase with sulbactam. The central intermediate can go on to form products, can transiently inhibit the enzyme in a quasi-reversible fashion, or can irreversibly inactivate the enzyme.

Beginning in the late 1980s, three -lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) have been used against serine enzymes, usually in combination with penicillins more susceptible to /1-lactamase hydrolysis. This therapeutic strategy has been effective over two decades. The following section provides a brief overview on various classes of -lactam-based inhibitors. 

Buynak et al. [53] synthesized several 6-(mercaptomethyl) penicillanates (9r and 9s, Table 1) that include both C-6 stereoisomers as well as the sulfide and sulfone oxidation states of the penam thiazolidine sulfur. Selected mercaptomethyl penicillanates inactivated both metallo- and serine /5-lactamases, and displayed synergism with piperacillin against various //-lactamase-producing strains, including metallo-/5-lactamase-producing P. aeruginosa strain. Compound 9r would be capable of bidentate chelation of zinc subsequent to enzymatic hydrolysis of the /5-lactam (Scheme 3). 

The discovery of the ethylidenecarbapenems, the asparenomycins, as naturally occurring /J-lactamase inactivators in the early 1980s was another striking point in /J-lactamase inhibitor research. The substituted exomethylene function in asparenomycins is a distinctive feature of this class of compounds, which many scientists recognized could be a key factor for /J-lactamase inhibition. The exo cyclic methylene is expected to increase the acylation ability, and form an a,/J-unsaturated ester of the active site serine residue as an acyl-enzyme complex. This ester will be similar in structure to the acyl-enzymes formed from clavulanate and sulfone fragmentation, and will be quite resistant to hydrolytic deacylation. Thus, the exocyclic methylene promotes acylation by the enzyme and subsequently represses deacylation. Based on... 

Other serine hydrolases such as cholinesterases, carboxylesterases, lipases, and fl-lactamases of classes A, C, and D have a hydrolytic mechanism similar to that of serine peptidases [25-27], The catalytic mechanism also involves an acylation and a deacylation step at a serine residue in the active center (see Fig. 3.3). All serine hydrolases have in common that they are inhibited by covalent attachment of diisopropyl phosphorofluoridate (3.2) to the catalytic serine residue. The catalytic site of esterases and lipases has been less extensively investigated than that of serine peptidases, but much evidence has accumulated that they also contain a catalytic triad composed of serine, histidine, and aspartate or glutamate (Table 3.1). 

Lactamases of classes A, C, and D are serine peptidases and as such have been discussed in Sect. 3.3. Class B /1-lactamases, in contrast, are met-allohydrolases. For example, a class B /1-lactamase isolated from Bacillus cereus was shown to contain two Zn-atoms per protein molecule, of which only one is essential for catalysis. Three histidine residues act as ligands for the first Zn2+ ion, and a fourth histidine contributes to the binding of the second Zn-atom [82] [83],... 

M. Galleni, J. Lamotte-Brasseur, X. Raquet, A. Dubus, D. Monnaie, J. R. Knox, J. M. Frere, The Enigmatic Catalytic Mechanism of Active-Site Serine /3-Lactamases , Bio-chem. Pharmacol. 1995,49, 1171-1178. 

Lactamases (EC 3.5.2.6) inactivate /3-lactam antibiotics by hydrolyzing the amide bond (Fig. 5.1, Pathway b). These enzymes are the most important ones in the bacterial defense against /3-lactam antibiotics [15]. On the basis of catalytic mechanism, /3-lactamases can be subdivided into two major groups, namely Zn2+-containing metalloproteins (class B), and active-serine enzymes, which are subdivided into classes A, C, and D based on their amino acid sequences (see Chapt. 2). The metallo-enzymes are produced by only a relatively small number of pathogenic strains, but represent a potential threat for the future. Indeed, they are able to hydrolyze efficiently carbape-nems, which generally escape the activity of the more common serine-/3-lac-tamases [16] [17]. At present, however, most of the resistance of bacteria to /3-lactam antibiotics is due to the activity of serine-/3-lactamases. These enzymes hydrolyze the /3-lactam moiety via an acyl-enzyme intermediate similar to that formed by transpeptidases. The difference in the catalytic pathways of the two enzymes is merely quantitative (Fig. 5.1, Pathways a and b). 

The /3-lactam structure can also react with active-serine hydrolases other than PBPs and /3-lactamases. It has been shown that appropriately substituted cephalosporins (e.g., 5.18) are potent mechanism-based inactivators of human leukocyte elastase (HLE, EC 3.4.21.37), a serine endopeptidase involved in the pathogenesis of pulmonary emphysema and other connective tissue diseases [57-60]. Subsequent work has demonstrated that substituted /3-lactams such as 5.19 or 5.20 are more stable HLE inhibitors and have improved potencies [61-63]. 

J. M. Ghuysen, Serine beta-Lactamases and Penicillin-Binding Proteins , Annu. Rev. Microbiol. 1991, 45, 37-67. 

A. Matagne, J. Lamotte-Brasseur, G. Dive, J. M. Frere, Interaction between Active Site-Serine /3-Lactamases and Compounds Bearing a Methoxy Side Chain on the a-Face of the /3-Lactam Ring Kinetic and Molecular Modelling , Biochem. J. 1993, 293, 607-611. 

N. Li, J. Rahil, M. E. Wright, R. F. Pratt, Structure-Activity Studies of the Inhibition of Serine beta-Lactamases by Phosphonate Monoesters , Bioorg. Med. Chem. 1997, 5, 1783-1788. 

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