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Structural consequences of the active site substitution Cys181 ⇒ Ser in metallo-β-lactamase from Bacteroides fragilis
Ist Teil von
Protein science, 1999-01, Vol.8 (1), p.249-252
Ort / Verlag
United States: Cambridge University Press
Erscheinungsjahr
1999
Link zum Volltext
Quelle
Electronic Journals Library
Beschreibungen/Notizen
The metallo-β-lactamases require divalent cations
such as zinc or cadmium for hydrolyzing the amide bond
of β-lactam antibiotics. The crystal structure of the
Zn2+-bound enzyme from Bacteroides fragilis
contains a binuclear zinc center in the active site. A
hydroxide, coordinated to both zinc atoms, is proposed
as the moiety that mounts the nucleophilic attack on the
carbonyl carbon atom of the β-lactam bond of the substrate.
It was previously reported that the replacement of the
active site Cys181 by a serine residue severely impaired
catalysis while atomic absorption measurements indicated
that binding of the two zinc ions remained intact. Contradicting
data emerge from recent mass spectrometry results, which
show that only a single zinc ion binds to the C181S metallo-β-lactamase.
In the current study, the C181S mutant enzyme was examined
at the atomic level by determining the crystal structure
at 2.6 Å resolution. The overall structure of the
mutant enzyme is the same as that of the wild-type enzyme.
At the mutation site, the side chain of Ser181 occupies
the same position as that of the side chain of Cys181 in
the wild-type protein. One zinc ion, Zn1, is present in
the crystal structure; however, the site of the second
zinc ion, Zn2 is unoccupied. A water molecule is associated
with Zn1, reminiscent of the hydroxide seen in the structure
of the wild-type enzyme but farther from the metal. The
position of the water molecule is off the plane of the
carboxylate group of Asp103; therefore, the water molecule
may be less nucleophilic than a water molecule which is
coplanar with the carboxylate group.