| Autor: |
Toney JH; Department of Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065-0900, USA. jeff_toney@merck.com, Hammond GG, Leiting B, Pryor KD, Wu JK, Cuca GC, Pompliano DL |
| Jazyk: |
angličtina |
| Zdroj: |
Analytical biochemistry [Anal Biochem] 1998 Jan 01; Vol. 255 (1), pp. 113-9. |
| DOI: |
10.1006/abio.1997.2458 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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