Soluble penicillin-binding protein 2a: beta-lactam binding and inhibition by non-beta-lactams using a 96-well format.
High level methicillin resistance in Staphylococcus aureus is dependent upon the acquisition of the mecA gene encoding penicillin-binding protein 2a (PBP2a). PBP2a is a member of a family of peptidoglycan biosynthetic enzymes involved in assembly of the cell wall in bacteria and is poorly inactivated by beta-lactam antibiotics. We describe a 96-well-filter binding assay using recombinant, soluble PBP2a which allows for kinetic measurement of penicillin binding. The deacylation rate constant for the PBP2a-penicillin G covalent complex was found to be 5.7 +/- 1.0 x 10(-5) s-1 at 30 degrees C (half-life of approximately 200 min). For the PBP2a acylation reaction, the value of K(m) (penicillin G) = 0.5 +/- 0.1 mM and kcat = 1 x 10(-3) s-1, which yields a second-order rate constant (kcat/K(m)) for inactivation of 2.0 M-1 s-1. Using this assay, several non-beta-lactam inhibitors including Cibacron blue have been found which exhibit IC50 values between 10 and 30 microM. The binding affinities of several carbapenems and beta-lactams correlated well between the filter binding assay described in this report and an electrophoretic assay for PBP2a using membranes prepared form methicillin-resistant S. aureus.
Toney
J H
JH
Department of Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065-0900, USA. jeff_toney@merck.com
Hammond
G G
GG
Leiting
B
B
Pryor
K D
KD
Wu
J K
JK
Cuca
G C
GC
Pompliano
D L
DL
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins; D001665:Binding Sites; D001667:Binding, Competitive; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods; D015780:Carbapenems / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011232:Chemical Precipitation; D004121:Dimethyl Sulfoxide; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006602:Hexosyltransferases; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism; D015378:Imipenem / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008722:Methods; D008851:Micropore Filters; D002267:Muramoylpentapeptide Carboxypeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010400:Penicillin G / Q000737:chemistry / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D012995:Solubility; D014227:Triazines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014316:Tritium
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015780:Carbapenems; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D007093:Imidazoles; C093180:L 695256; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D014227:Triazines; D014316:Tritium; C012842:Cibacron Blue F 3GA; D015378:Imipenem; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D010400:Penicillin G; D004121:Dimethyl Sulfoxide