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J Biol Chem. 2014 Feb 28;289(9):6152-64. doi: 10.1074/jbc.M113.533562. Epub 2014 Jan 17.

The different inhibition mechanisms of OXA-1 and OXA-24 β-lactamases are determined by the stability of active site carboxylated lysine.

Author information

1
From the Departments of Biochemistry.

Abstract

The catalytic efficiency of class D β-lactamases depends critically on an unusual carboxylated lysine as the general base residue for both the acylation and deacylation steps of the enzyme. Microbiological and biochemical studies on the class D β-lactamases OXA-1 and OXA-24 showed that the two enzymes behave differently when reacting with two 6-methylidene penems (penem 1 and penem 3): the penems are good inhibitors of OXA-1 but act more like substrates for OXA-24. UV difference and Raman spectroscopy revealed that the respective reaction mechanisms are different. The penems form an unusual intermediate, a 1,4-thiazepine derivative in OXA-1, and undergo deacylation followed by the decarboxylation of Lys-70, rendering OXA-1 inactive. This inactivation could not be reversed by the addition of 100 mM NaHCO3. In OXA-24, under mild conditions (enzyme:inhibitor = 1:4), only hydrolyzed products were detected, and the enzyme remained active. However, under harsh conditions (enzyme:inhibitor = 1:2000), OXA-24 was inhibited via decarboxylation of Lys-84; however, the enzyme could be reactivated by the addition of 100 mM NaHCO3. We conclude that OXA-24 not only decarboxylates with difficulty but also recarboxylates with ease; in contrast, OXA-1 decarboxylates easily but recarboxylates with difficulty. Structural analysis of the active site indicates that a crystallographic water molecule may play an important role in carboxylation in OXA-24 (an analogous water molecule is not found in OXA-1), supporting the suggestion that a water molecule in the active site of OXA-24 can lower the energy barrier for carboxylation significantly.

KEYWORDS:

Carboxylated Lysine; Crystallography; Enzyme Mechanisms; OXA-1; OXA-24; Penems; Physical Methods; Raman Spectroscopy; UV Spectroscopy

PMID:
24443569
PMCID:
PMC3937681
DOI:
10.1074/jbc.M113.533562
[Indexed for MEDLINE]
Free PMC Article

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