The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme (original) (raw)
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Recognition and Resistance in TEM β-Lactamase †
Biochemistry, 2003
Developing antimicrobials that are less likely to engender resistance has become an important design criterion as more and more drugs fall victim to resistance mutations. One hypothesis is that the more closely an inhibitor resembles a substrate, the more difficult it will be to develop resistant mutations that can at once disfavor the inhibitor and still recognize the substrate. To investigate this hypothesis, 10 transition-state analogues, of greater or lesser similarity to substrates, were tested for inhibition of TEM-1 -lactamase, the most widespread resistance enzyme to penicillin antibiotics. The inhibitors were also tested against four characteristic mutant enzymes: TEM-30, TEM-32, TEM-52, and TEM-64. The inhibitor most similar to the substrate, compound 10, was the most potent inhibitor of the WT enzyme, with a K i value of 64 nM. Conversely, compound 10 was the most susceptible to the TEM-30 (R244S) mutant, for which inhibition dropped by over 100-fold. The other inhibitors were relatively impervious to the TEM-30 mutant enzyme. To understand recognition and resistance to these transition-state analogues, the structures of four of these inhibitors in complex with TEM-1 were determined by X-ray crystallography. These structures suggest a structural basis for distinguishing inhibitors that mimic the acylation transition state and those that mimic the deacylation transition state; they also suggest how TEM-30 reduces the affinity of compound 10. In cell culture, this inhibitor reversed the resistance of bacteria to ampicillin, reducing minimum inhibitory concentrations of this penicillin by between 4-and 64-fold, depending on the strain of bacteria. Notwithstanding this activity, the resistance of TEM-30, which is already extant in the clinic, suggests that there can be resistance liabilities with substrate-based design.
β-Lactamases: A Focus on Current Challenges
Cold Spring Harbor Perspectives in Medicine, 2016
b-Lactamases, the enzymes that hydrolyze b-lactam antibiotics, remain the greatest threat to the usage of these agents. In this review, the mechanism of hydrolysis is discussed for both those enzymes that use serine at the active site and those that require divalent zinc ions for hydrolysis. The b-lactamases now include .2000 unique, naturally occurring amino acid sequences. Some of the clinically most important of these are the class A penicillinases, the extended-spectrum b-lactamases (ESBLs), the AmpC cephalosporinases, and the carbapenem-hydrolyzing enzymes in both the serine and metalloenzyme groups. Because of the versatility of these enzymes to evolve as new b-lactams are used therapeutically, new approaches to antimicrobial therapy may be required.
The diversity of the catalytic properties of class A β-lactamases
Biochemical Journal, 1990
The catalytic properties of four class A beta-lactamases were studied with 24 different substrates. They exhibit a wide range of variation. Similarly, the amino acid sequences are also quite different. However, no relationships were found between the sequence similarities and the substrate profiles. Lags and bursts were observed with various compounds containing a large sterically hindered side chain. As a group, the enzymes could be distinguished from the class C beta-lactamases on the basis of the kappa cat. values for several substrates, particularly oxacillin, cloxacillin and carbenicillin. Surprisingly, that distinction was impossible with the kappa cat./Km values, which represent the rates of acylation of the active-site serine residue by the beta-lactam. For several cephalosporin substrates (e.g. cefuroxime and cefotaxime) class A enzymes consistently exhibited higher kappa cat. values than class C enzymes, thus belying the usual distinction between ‘penicillinases’ and ‘ceph...
FEMS Microbiology Letters, 2000
The plasmid-mediated TEM-I and TEM-2 p-lactamases are the most commonly encountered among Gram-negative bacteria. They belong to molecular class A, and differ by one amino acid at position 39 : TEM-1 have a glutamine and TEM-2 a lysine. Kinetic parameters (& and I&) and catalytic efftciency (k,,JK,,,) of TEM-1 and TEM-2 P-lactamases are slightly, but significantly different. For all antibiotics except methicillin and cefazolin, the catalytic efficiency values of TEM-2 are clearly greater than that of TEM-1. Molecular modelling of TEM-2, when compared to that of TEM-1, showed an additional ionic bond between Lys-39 and Glu-281.
Past and Present Perspectives on β-Lactamases
Antimicrobial Agents and Chemotherapy, 2018
β-Lactamases, the major resistance determinant for β-lactam antibiotics in Gram-negative bacteria, are ancient enzymes whose origins can be traced back millions of years ago. These well-studied enzymes, currently numbering almost 2,800 unique proteins, initially emerged from environmental sources, most likely to protect a producing bacterium from attack by naturally occurring β-lactams. Their ancestors were presumably penicillin-binding proteins that share sequence homology with β-lactamases possessing an active-site serine. Metallo-β-lactamases also exist, with one or two catalytically functional zinc ions. Although penicillinases in Gram-positive bacteria were reported shortly after penicillin was introduced clinically, transmissible β-lactamases that could hydrolyze recently approved cephalosporins, monobactams, and carbapenems later became important in Gram-negative pathogens. Nomenclature is based on one of two major systems. Originally, functional classifications were used, ba...
Drifted catalytic properties of β-lactamases due to unconstrained use of antibiotics
Context: Antibiotic resistance is an old problem with new face as the rate of infections due to multidrug resistant bacteria is increasing everyday and the number of new antibiotics to overwhelm the problem is becoming smaller. Major mechanism beneath this growing resistance is concomitant with the changes in β-lactamases catalytic activity and its functional enhancement. Objectives: In β-lactamases secreting clinical isolates at least 10% are extended-spectrum β-lactamases (ESBL) that are not even treatable with β-lactamases inhibitor like clavulanic acids. This implies that the catalytic domains of β-lactamases have been mutated towards higher pathogenicity. The aim of the present study is to define the changes in β-lactamases catalytic efficiency against β-lactam antibiotics and its inhibitors. Materials and Methods: In this research work we have used multiple drug resistant (MDR) strains from surgical site of infections. A rapid method was used for specific detection of bacterial β-lactamases that uses β-lactam antibiotics as substrates. In this, the end products (open beta-lactam ring forms) generated after separately incubating substrates with β-lactamases producing strains. Those end products of antibiotics were highly fluorescent after specific treatment and could be analyzed visually under long-wave UV lamp for efficiency. Results: β-lactamases secreting strains are variably capable of defending β-lactam antibiotics. Interestingly, one of the E. coli strain secretes ESBL, this means that the strain is resistant against clavulanic acid. However, the most fascinating fact of the finding is that ideally the β-lactamases supposed to hydrolyze Penicillin by default but in our isolates, β-lactamases are not able to hydrolyze penicillin instead they hydrolyze amoxicillin, a derivative which replaced clinical use of penicillin. In addition to that we have identified the presence of New Delhi Metalo-betalactamase in one of the clinical isolates. Conclusion: Rate of evolution in microbes is very high. Thus we presume that some of the amino acids in the functional domain of β-lactamases have been changed respective to extinct use of penicillin whereas it is effective against clinically used other beta lactam antibiotics.