Modeling Study on a Hydrolytic Mechanism of Class A β-Lactamases

Abstract

Comparison of the hydrogen-bond networks at the active site in the crystallographic structures reported for class A beta-lactamases revealed an importance of a switch of the hydrogen-bond network for the catalytic process. Taking account of the conformational mobility of the Lys73 residue, we have constructed putative complex models for beta-lactam antibiotics and the enzymes in the multistep hydrolysis which consists of a Michaelis complex, an acyl-enzyme, and a tetrahedral oxyanion for deacylation. In the acylation, the C3 carboxylate of penicillin derivatives would participate in activation of the Ser130 hydroxyl group and then the oxyanion of the Ser130 residue would deprotonate the ammonium group of the Lys73 residue which will act as a general base for activation of the Ser70 residue. In the deacylation, the deacylating water molecule would be accommodated during a conformational change of the acyl moiety without a structural change of the active-site residues and the unprotonated N4 atom of the penicillins would act as a general base to activate the water molecule. This catalytic process provided a new account for the stability of the acyl-enzyme complexes. This substrate-assisted mechanism would also be extended to a hydrolytic mechanism of class C enzymes.

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

“…It has been proposed that SAC can be observed in the catalytic mechanisms of many different types of enzymes 32) as well as class A b-lactamase. The SAC found in this study is similar to the mechanism proposed by Ishiguro and Imajo 15) because the deprotonation of Lys73 is induced by the C3-carboxyl group in both Ishiguro's mechanism and ours. Furthermore, Zawadzke et al suggested that the pK a of Lys73 was dramatically reduced upon substrate binding, 33) which is also compatible with our mechanism.…”

“…Ishiguro et al also discussed the importance of the hydrogen bond network based on the results of their molecular mechanics calculations. 15) Accordingly, we investigated the whole mechanism of the deacylation reaction by theoretical calculation.…”

“…1 (c) to (d)). The acylation mechanism has been examined in many experimental [6][7][8][9][10][11][12][13][14] and theoretical works, 11,[15][16][17][18][19][20][21][22] and acylation is common to serine-b-lactamase and penicillin-binding protein (PBP). Deacylation, on the other hand, is only seen in serine-b-lactamase.…”

Role of a Mutation at Position 167 of CTX-M-19 in Ceftazidime Hydrolysis

“…The side chain hydroxyl group of Ser237 also had a different conformation to preferentially form a hydrogen bond with the sulfonate group of aztreonam, and this interaction interfered with the interaction between the sulfonate group and Ser130. Since the electronic interaction of the Ser130 hydroxyl group a the negative group such as sulfonate group in aztreonam would facilitate the enzyme reaction (12), the preferential interaction between the sulfonate group and Ser130 of CTX-M-18 would lead to effective hydrolysis of aztreonam, as observed in the hydrolysis of aztreonam by CTX-M-18.…”

“…This may be compatible with the finding that both enzymes have a similar affinity for cefotaxime. Further inspection of the complex models, Ser130 in the CTX-M-18 model had a hydrogen bond with Lys73, which is assumed to be a general base for activating the hydroxyl group of Ser70 for acylation (12), whereas Ser130 in the CTX-M-19 model was not located in a hydrogen bond distance to Lys73.…”