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

✍ Scribed by 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 Jr.; David Tew; Igor I. Goryanin

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 589 KB
• Volume: 95
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/bit.20772

✦ Synopsis

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. ß 2006 Wiley Periodicals, Inc.