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Biotechnol Bioeng. 2006 Nov 5;95(4):560-73.

The kinetic model of the shikimate pathway as a tool to optimize enzyme assays for high-throughput screening.

Author information

1
Assay Methodology Development, GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, United Kingdom.

Abstract

Four-enzyme section of the shikimate pathway (Aro B, D, E, and K) of Streptococcus pneumoniae has been studied. Kinetic properties of the individual enzymes and three- and four-enzyme linked reactions have been characterized in vitro. On the basis of the data measured in spectrophotometric and LC-MS experiments, kinetic mechanisms of the enzymes have been suggested and all kinetic parameters have been identified. Kinetic models for these three- and four-enzyme sections of the shikimate pathway have been constructed and validated. The model of the four-enzyme section of shikimate pathway has been employed to design an inhibition-sensitive reconstituted pathway for a high-throughput screening effort on the shikimate pathway. It was demonstrated that using the model it was possible to optimize this reconstituted pathway in such a way to provide equal sensitivity of the enzymes to inhibition.

PMID:
16921527
DOI:
10.1002/bit.20772
[Indexed for MEDLINE]

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