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

Michael Noble, Yugesh Sinha, Aleksey Kolupaev, Oleg Demin, David Earnshaw, Frank Tobin, Joshua West, John D. Martin, Chunyan Qiu, Wu-Schyong Liu, Walter E. DeWolf, David Tew, Igor Goryanin

Research output: Contribution to journalArticlepeer-review

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. © 2006 Wiley Periodicals, Inc.
Original languageEnglish
Pages (from-to)560-571
Number of pages12
JournalBiotechnology and Bioengineering
Volume95
Issue number4
DOIs
Publication statusPublished - 12 Aug 2006

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