L.W.Tremblay
et al.
(2010).
Biochemical and structural characterization of Mycobacterium tuberculosis beta-lactamase with the carbapenems ertapenem and doripenem.
Biochemistry,
49,
3766-3773.
PubMed id: 20353175
Biochemical and structural characterization of Mycobacterium tuberculosis beta-lactamase with the carbapenems ertapenem and doripenem.
L.W.Tremblay,
F.Fan,
J.S.Blanchard.
ABSTRACT
Despite the enormous success of beta-lactams as broad-spectrum antibacterials,
they have never been widely used for the treatment of tuberculosis (TB) due to
intrinsic resistance that is caused by the presence of a chromosomally encoded
gene (blaC) in Mycobacterium tuberculosis. Our previous studies of TB BlaC
revealed that this enzyme is an extremely broad-spectrum beta-lactamase
hydrolyzing all beta-lactam classes. Carbapenems are slow substrates that
acylate the enzyme but are only slowly deacylated and can therefore act also as
potent inhibitors of BlaC. We conducted the in vitro characterization of
doripenem and ertapenem with BlaC. A steady-state kinetic burst was observed
with both compounds with magnitudes proportional to the concentration of BlaC
used. The results provide apparent K(m) and k(cat) values of 0.18 microM and
0.016 min(-1) for doripenem and 0.18 microM and 0.017 min(-1) for ertapenem,
respectively. FTICR mass spectrometry demonstrated that the doripenem and
ertapenem acyl-enzyme complexes remain stable over a time period of 90 min. The
BlaC-doripenem covalent complex obtained after a 90 min soak was determined to
2.2 A, while the BlaC-ertapenem complex obtained after a 90 min soak was
determined to 2.0 A. The 1.3 A diffraction data from a 10 min ertapenem-soaked
crystal revealed an isomerization occurring in the BlaC-ertapenem adduct in
which the original Delta(2)-pyrroline ring was tautomerized to generate the
Delta(1)-pyrroline ring. The isomerization leads to the flipping of the
carbapenem hydroxyethyl group to hydrogen bond to carboxyl O2 of Glu166. The
hydroxyethyl flip results in both the decreased basicity of Glu166 and a
significant increase in the distance between carboxyl O2 of Glu166 and the
catalytic water molecule, slowing hydrolysis.
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