Deacylation kinetics analysis of Streptococcus pneumoniae penicillin-binding protein 2x mutants resistant to beta-lactam antibiotics using electrospray ionization- mass spectrometry.
Penicillin-binding proteins (PBPs) catalyze the transpeptidase reaction involved in peptidoglycan synthesis and are covalently inhibited by the beta-lactam antibiotics. In a previous work we have focused on acylation efficiency measurements of various Streptococcus pneumoniae PBP2x* mutants to study the molecular determinants of resistance to beta-lactams. In the present paper we have developed a method to improve an accurate determination of the deacylation rate constant using electrospray ionization-mass spectrometry. This method is adaptable to the analysis of deacylation of any beta-lactam. Compared to the fluorographic technique, the ESI-MS method is insensitive to variations in the concentration of functional proteins and is therefore more reliable. We have established that the resistance of PBPs to beta-lactams is mostly due to a decrease of the acylation efficiency with only marginal effects on the deacylation rates.
Di Guilmi
A M
AM
Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale Jean-Pierre Ebel (CEA/CNRS), 41 Rue Jules Horowitz, Grenoble, France.
Mouz
N
N
Pétillot
Y
Y
Forest
E
E
Dideberg
O
O
Vernet
T
T
D000215:Acylation; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004352:Drug Resistance, Microbial / Q000235:genetics; D006602:Hexosyltransferases; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D013296:Streptococcus pneumoniae / Q000235:genetics*; D047090:beta-Lactams
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase