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Biochem. J. (2004) 383
(551–559) (Printed in Great Britain)
Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay
Sheraz GUL* 1, Richard BROWN†, Earl MAY‡, Marie MAZZULLA‡, Martin G. SMYTH§, Colin BERRY  , Andrew MORBY and David J. POWELL* *Assay Development and Compound Profiling, GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park (North), Third Avenue, Harlow, Essex CM19 4AW, U.K., †Molecular Light Technology Research Ltd, 5 Chiltern Close, Cardiff Industrial Park, Cardiff CF14 5DL, Wales, U.K., ‡MMPD CEDD, GlaxoSmithKline Pharmaceuticals, 1250 S. Collegeville Road, P.O. Box 5089, Collegeville, PA 19426, U.S.A., §Gene Expression and Protein Biochemistry, GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park (North), Third Avenue, Harlow, Essex CM19 4AW, U.K., and School of Biosciences, Cardiff University, Museum Avenue, PO Box 911, Cardiff CF10 3US, Wales, U.K.
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 Km values for NAD+ (2.75±0.1 µM) and the acridinium-ester-labelled DNA substrate (2.5±0.2 nM). A study of the pH-dependencies of kcat, Km and kcat/Km has revealed values of kinetically influential ionizations within the enzyme–substrate complexes (kcat) and free enzyme (kcat/Km). In each case, the curves were shown to be composed of one kinetically influential ionization, for kcat, pKa=6.6±0.1 and kcat/Km, pKa=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 µM for doxorubicin and 1.40±0.07 µM 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 µl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.
Key words: acridinium ester label, chemiluminescent hybridization protection assay, high-throughput screening, kinetic characterization, lead discovery, Staphylococcus aureus DNA ligase.
Abbreviations used: AE, acridinium ester; DTT, dithiothreitol; HPA, hybridization protection assay; HTS, high-throughput screening; NHS, N-hydroxysuccinamide; RLU, relative light units.
1To whom correspondence should be addressed (email Sheraz_2_Gul@gsk.com).
Received 8 January 2004/21 July 2004; accepted 29 July 2004
Published as BJ Immediate Publication 29 July 2004, DOI 10.1042/BJ20040054
The Biochemical Society, London ©2004
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