Purification, properties, and kinetics of enzymatic acylation with beta-lactams of soluble penicillin-binding protein 2a. A major factor in methicillin-resistant Staphylococcus aureus.
Intrinsic resistance toward beta-lactams in methicillin-resistant Staphylococcus aureus strains, a major source of nosocomial infections, is believed to be caused mainly by penicillin-binding protein 2a (PBP2a). This protein resembles other penicillin-binding proteins that are involved in bacterial cell wall biosynthesis and are the targets of active site acylation by beta-lactam antibiotics. PBP2a, however, presumably remains active at therapeutic concentrations of beta-lactams. In this paper, we describe a three-step purification of a soluble form of PBP2a (PBP2a') to apparent homogeneity using anion-and cation-exchange, and dye-ligand affinity chromatographies. Purified PBP2a' was a 74-kDa monomeric protein that appeared to be folded. The protein was evaluated for its enzymatic acylation with beta-lactams initially by fluorescence quenching and then kinetically by radioactive labeling. Using a modified 125I-labeled penicillin V-acylation assay, the apparent Km of PBP2a' for penicillin V was 1.2 mM. Three other beta-lactams, each of which exhibited significant fluorescence quenching, acted as strong competitive inhibitors of penicillin V with apparent Ki values of 123.4, 36.1, and 12.4 microM, respectively. By a new beta-lactam preincubation analysis, these compounds could function as substrates with similar Km values. Also, the acylation rates of different beta-lactams could be readily ascertained. The enzymatic acylation data substantiate the major causative role of PBP2a in the bacterial resistance. The quantitative radioactive acylation assays are potentially useful in screening for a potent inhibitor of the enzyme.
Roychoudhury
S
S
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285.
Dotzlaf
J E
JE
Ghag
S
S
Yeh
W K
WK
D000215:Acylation; D000596:Amino Acids / Q000032:analysis; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006602:Hexosyltransferases; D007700:Kinetics; D016106:Methicillin Resistance / Q000502:physiology*; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010404:Penicillin V / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D010458:Peptidyl Transferases; D010957:Plasmids; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000201:enzymology*; D013997:Time Factors
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D010404:Penicillin V