The kinetic model of the shikimate pathway as a tool to optimize enzyme assays for high-throughput screening.
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.
Noble
Michael
M
Assay Methodology Development, GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, United Kingdom.
Sinha
Yugesh
Y
Kolupaev
Aleksey
A
Demin
Oleg
O
Earnshaw
David
D
Tobin
Frank
F
West
Joshua
J
Martin
John D
JD
Qiu
Chunyan
C
Liu
Wu-Schyong
WS
DeWolf
Walter E
WE
Tew
David
D
Goryanin
Igor I
II
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways; D015964:Gene Expression Regulation, Bacterial; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008967:Molecular Biology / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D013296:Streptococcus pneumoniae / Q000201:enzymology*
D000429:Alcohol Oxidoreductases; C022641:Shikimate dehydrogenase; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase; C020001:3-dehydroquinate synthetase; D019761:Phosphorus-Oxygen Lyases