␤-Lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to ␤-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins, and crystal structures demonstrate that the side-chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys-73) in the TEM-1 ␤-lactamase from Escherichia coli. These techniques included perturbation of the pK a of Lys-73 by the study of the ␥-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance, and by computational treatment using the thermodynamic integration method. All three methods indicated that the pK a of Lys-73 of this enzyme is attenuated to 8.0 -8.5. It is argued herein that the unique ground-state ion pair of Glu-166 and Lys-73 of class A ␤-lactamases has actually raised the pK a of the active site lysine to 8.0 -8.5 from that of the parental penicillin-binding protein. Whereas we cannot rule out that Glu-166 might activate the active site water, which in turn promotes Ser-70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu-166 and unprotonated Lys-73. As such, unprotonated Lys-73 could promote serine for acylation, a process that should be shared among all active-site serine ␤-lactamases and penicillin-binding proteins.A number of enzymes have evolved a catalytic strategy that depends on a transient acylation of an active site serine. The catalytic serine residue in these enzymes is followed by a lysine three residues toward the carboxyl termini of the proteins (i.e. . . . Ser-X-X-Lys . . . ). This sequence motif is seen in serinedependent ␤-lactamases and penicillin-binding proteins (PBPs 1 ), of which several hundred members are known. The catalytic implication of this Ser-X-X-Lys sequence motif for ␤-lactamases is debated in the literature, but the role of these residues in catalysis is likely to be general for the large group of proteins that share this sequence.␤-Lactamases are bacterial resistance enzymes to ␤-lactam antibiotics, which include penicillins and cephalosporins. Members of the class A ␤-lactamases are the most common among pathogenic bacteria. These enzymes undergo acylation and deacylation at Ser-70 during substrate turnover (1, 2). The process of deacylation of the acyl-enzyme intermediate is best understood. Glu-166 is the active-site general base that promotes a water molecule in the deacylation step (3-5). On the other hand, how the active-site serine experiences ...

β-Lactamases of classes A and C are believed to have evolved from bacterial enzymes involved in biosynthesis of the peptidoglycan, the so-called penicillin-binding proteins. All these enzymes undergo acylation at an active-site serine by β-lactam antibiotics as a common feature. However, the fate of the acyl-enzyme species is different for β-lactamases and penicillin-binding proteins; deacylation is rapid for the former, whereas it is slow for the latter. It is believed that the acquisition of the ability to deacylate the acyl-enzyme intermediate led to the evolution of β-lactamase activity, which is indispensable for the survival of bacteria in the face of challenge by β-lactam antibiotics. The mechanisms of deacylation of acyl-enzyme intermediates for β-lactamases are examined as a means to investigate structural factors in evolutionary descendency of classes A and C of β-lactamases from penicillin-binding proteins. It is known that in class A β-lactamases the hydrolytic water approaches the acyl-enzyme intermediate from the α-face, a process which is promoted by Glu-166 of these enzymes. An approach from the β-face for class C β-lactamase has been proposed. The process of activation of the hydrolytic water is not entirely understood at the present for these enzymes. Two compounds, p-nitrophenyl (2R,5R)-5-prolylacetate (2) and p-nitrophenyl (1S,3S)-3-carboxycyclopentylacetate (3), were synthesized as mechanistic probes to explore whether the hydrolytic water molecule actually approaches the acyl-enzyme species from the β-face and to investigate a notion that the ring amine at the acyl-enzyme intermediate may promote the hydrolytic reaction. Compound 2 acylates the active site serine of the Q908R β-lactamase (a class C enzyme), and the intermediate undergoes deacylation. On the other hand, compound 3 only acylates the active site, and not having the requisite amine in its structure, the intermediate resists deacylation. Both compounds serve as substrates for the class A TEM-1 β-lactamase, as they were expected, since the approach of the hydrolytic water molecule is from the α-face in this enzyme and is not promoted by the substrate itself. We conclude that substrate-assisted catalysis applies for the class C β-lactamases. On the basis of the evidence discussed, the knowledge of the crystal structures for the classes A and C of β-lactamases and the Steptomyces R61 DD-peptidase/transpeptidase (a PBP), it is proposed herein that evolution of classes A and C of β-lactamases from a primordial penicillin-binding protein should have been independent events; hence, the process does not represent a linear descendency of one β-lactamase from the other.

The clinical use of beta-lactam antibiotics combined with beta-lactamase inactivators, such as clavulanate, has resulted in selection of beta-lactamases that are insensitive to inactivation by these molecules. Therefore, therapeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme. The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 variants are designated as inhibitor-resistant TEM ("IRT") enzymes. Three mutations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution. In this study, we have characterized the kinetic properties of the inhibition process of the wild-type TEM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam, and we report the X-ray structure for the mutant variant at 2.3 A resolution. The changes in kinetic parameters for the interactions of the inhibitors with the wild-type and the mutant enzymes were more pronounced for clavulanate, and relatively inconsequential for sulbactam and tazobactam. The structure of the Asn276Asp mutant enzyme revealed a significant movement of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate. A water molecule critical for the inactivation chemistry by clavulanate, which is observed in the wild-type enzyme structure, is not present in the crystal structure of the mutant variant. Such structural changes favor the turnover process over the inactivation chemistry for clavulanate, with profound phenotypic consequences. The report herein represents the best studied example of inhibitor-resistant beta-lactamases.