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Binding and Hydrolysis of Ampicillin in the Active Site of a Zinc Lactamase

Published

Author(s)

Morris Krauss, N. Gresh, J Antony

Abstract

Binding and hydrolysis of ampicillin is described in a model active site derived from di-nuclear B.fragilis zinc lactamase. The protein binding site consists of the two zinc cations bound with a bridging hydroxide and ligands from the first-shell residues, conserved residues near the zinc site, and the moveable loop of residues from numbers 43-53. The model active site consists of the first-shell residues, the conserved residues, and glu45 and glu47 from the moveable loop. Ampicillin is primarily located in the active site by the binding of the ring carboxylate to the ammonium of conserved lysine 184 when water bound to Zn2 in the active site is retained. A comparable strong salt-bridge is formed between the ammonium of the ampicillin zwitterion and glu45 on the flexible loop that moves mostly as a unit at least 10 to complete the binding site. The zwitterion character of this antibiotic influences the binding arrangement and ultimate reaction path. Classical molecular mechanics minimization in the absence of water other than the Zn2 bound water determined a number of docking conformations. One of the structures with strong interactions to glu45 and glu47 was chosen to calculate the reaction path for binding reactant, intermediates, and product for the initial hydrolysis reaction. Water is added to solvate the initial reactant structure and the reaction path was calculated quantum mechanically within a model chosen from the molecular mechanics structure. Two waters were found in a productive conformation for hydrolysis, the water bound to Zn2 and water bound to the ampicillin carboxylate. Intermediates on the two reaction paths are both stabilized by interaction with the zinc cations but different zinc atoms are used in the two paths suggesting that neither zinc is specially chosen for a catalytic role. The hydroxylated ampicillin intermediate is bound directly to Zn1 in one reaction path and to Zn2 in the product of the second reaction path. Within this model the entire active site is utilized for both binding and catalysis in the case of ampicillin. Strong polar hydrogen-bonds are found to the substrate, the waters in the active site, and the residue ligands present in the active site. Autocatalysis or assistance in water activation by the carboxylate of the antibiotic is found and likely to be general. The proton abstracted from the water can park to a number of anionic or polar atom sites in the active site leading to a range of intermediates. The ring C-N bond does not break with prior protonation of the nitrogen but requires attack of the hydroxide at the carbonyl carbon either prior to proton binding or concurrently. This study provides insight into a wider variety of antibiotic docking and shows that more than one reaction path is possible within the highly ionic active site of a bi-metallic lactamase.
Citation
Journal of Physical Chemistry B
Volume
107
Issue
5

Keywords

ampicillin, antibiotic resistance, reaction paths, two-zinc reactive mechanism, zinc lactamase

Citation

Krauss, M. , Gresh, N. and Antony, J. (2003), Binding and Hydrolysis of Ampicillin in the Active Site of a Zinc Lactamase, Journal of Physical Chemistry B (Accessed April 24, 2024)
Created February 6, 2003, Updated February 19, 2017