IMP-1 metallo-beta-lactamase: effect of chelators and assessment of metal requirement by electrospray mass spectrometry.
Metallo-beta-lactamases have attracted considerable attention due to their role in microbial resistance to beta-lactam antibiotics. IMP-1, the binuclear Zn-dependent beta-lactamase produced by Pseudomonas aeruginosa and other microorganisms, is of particular interest in view of its increasing prevalence. An examination of the susceptibility of IMP-1 to inactivation by six different divalent metal ion chelators has revealed that all except Zincon cause inhibition by forming a complex with the holoenzyme. Exposure of the enzyme to dipicolinic acid (DPA), the most potent inhibitor, results in the production of the mononuclear Zn form of the protein as determined by electrospray ionization mass spectrometry (ESI-MS) under nondenaturing conditions. This mononuclear Zn species was found to be catalytically competent. Studies with the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) show that the two zinc centers in IMP-1 differ in their accessibility, a feature that could be overcome in the presence of guanidine hydrochloride (GdnHCl, 1.5 M).
Siemann
Stefan
S
Department of Chemistry, University of Waterloo, 200 University Ave. W, Waterloo, ON, Canada N2L 3G1.
Brewer
Dyanne
D
Clarke
Anthony J
AJ
Dmitrienko
Gary I
GI
Lajoie
Gilles
G
Viswanatha
Thammaiah
T
D002384:Catalysis / Q000187:drug effects; D002614:Chelating Agents / Q000737:chemistry* / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D019791:Guanidine / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D012118:Resorcinols / Q000737:chemistry / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D015032:Zinc / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry* / Q000187:drug effects*
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D012118:Resorcinols; C409201:beta-lactamase IMP-1; D001618:beta-Lactamases; D015032:Zinc; D019791:Guanidine; C028398:4-(2-pyridylazo)resorcinol