Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay
| Autor: | Colin Berry, Sheraz Gul, Marie J. Mazzulla, Earl May, Martin G. Smyth, Richard Charles Brown, Andrew P. Morby, David J. Powell |
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| Rok vydání: | 2004 |
| Předmět: |
DNA
Bacterial Staphylococcus aureus Hybridization Protection Assay DNA Ligases Oligonucleotides Biology Biochemistry Catalysis Substrate Specificity chemistry.chemical_compound Enzyme kinetics Cloning Molecular Molecular Biology chemistry.chemical_classification DNA ligase Staining and Labeling Hydrolysis DNA replication Nucleic Acid Hybridization Cell Biology Kinetics Enzyme chemistry Research Design Prokaryotic DNA replication Luminescent Measurements Acridines DNA Research Article |
| Zdroj: | Biochemical Journal. 383:551-559 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20040054 |
| Popis: | DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development. |
| Databáze: | OpenAIRE |
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