Studies on ternary metallo-beta lactamase-inhibitor complexes using electrospray ionization mass spectrometry.
Metallo-beta-lactamases (MBLs) are targets for medicinal chemistry as they mediate bacterial resistance to beta-lactam antibiotics. Electrospray-ionization mass spectrometry (ESI-MS) was used to study the inhibition by a set of mercaptocarboxylates of two representative MBLs with different optimal metal stoichiometries for catalysis. BcII is a dizinc MBL (Class B1), whilst the CphA MBL (Class B2) exhibits highest activity with a single zinc ion in the active site. Experimental parameters for the detection of the metallo-enzyme and the metallo-enzyme-inhibitor complexes were evaluated and optimized. Following investigations on the stoichiometry of metal binding, the affinity of the inhibitors was investigated by measuring the relative abundance of the complex compared to the metalloprotein. The results for the BcII enzyme were in general agreement with solution assays and demonstrated that the inhibitors bind to the dizinc form of the BcII enzyme. The results for the CphA(ZnII) complex unexpectedly revealed an increased affinity for the binding of a second metal ion in the presence of thiomandelic acid. The results demonstrate that direct ESI-MS analysis of enzyme:inhibitor complexes is a viable method for screening inhibitors and for the rapid assay of the enzyme:metal:inhibitor ratios.
Selevsek
Nathalie
N
Department of Biochemical Engineering, Saarland University, Saarbrücken, Germany.
Tholey
Andreas
A
Heinzle
Elmar
E
Liénard
Benoît M R
BM
Oldham
Neil J
NJ
Schofield
Christopher J
CJ
Heinz
Uwe
U
Adolph
Hans-Werner
HW
Frère
Jean-Marie
JM
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D015195:Drug Design; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry*; D008670:Metals; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015032:Zinc / Q000737:chemistry*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008670:Metals; D065093:beta-Lactamase Inhibitors; C455979:beta-lactamase BcII; D001618:beta-Lactamases; C082063:cphA protein, Aeromonas hydrophila; D015032:Zinc