title: A pH-based high-throughput assay for transketolase: fingerprinting of substrate tolerance and quantitative kinetics.
abstract: A pH-based high-throughput assay method has been developed for the rapid and reliable measurement of transketolase (TK) activity. The method is based on the decarboxylation of lithium hydroxypyruvate (HPA) as a hydroxyacetyl donor with an aldehyde acceptor, using phenol red as the pH indicator. Upon release of carbon dioxide from HPA, the pH increase in the reaction mixture can be determined photometrically by the color change of the pH indicator. At low buffer concentration (2 mM triethanolamine, pH 7.5), the method is highly sensitive and allows continuous monitoring, for quantitative determination of the kinetic parameters. By using this method, the substrate specificities of the TK enzymes from Escherichia coli and Saccharomyces cerevisiae, as well as two active-site-modified variants of the E. coli TK (D469E, H26Y) were evaluated against a panel of substrate analogues; specific activities and kinetic constants could be rapidly determined. Substrate quality indicated by assay determination was substantiated with novel TK applications by using achiral 3-hydroxypropanal and 4-hydroxybutanal for preparative synthesis of chiral deoxyketose-type products. Determination of ee for the latter could be performed by chiral GC analysis, with an unambiguous correlation of the absolute configuration from rotation data. This pH-based assay method is broadly applicable and allows rapid, sensitive, and reliable screening of the substrate tolerance of known TK enzymes and variants obtained from directed evolution.
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Petersenstrasse 22, 64287 Darmstadt, Germany.
authors.forename: Dong
authors.initials: D
authors.lastname: Yi
authors.forename: Titu
authors.initials: T
authors.lastname: Devamani
authors.forename: Juliane
authors.initials: J
authors.lastname: Abdoul-Zabar
authors.forename: Franck
authors.initials: F
authors.lastname: Charmantray
authors.forename: Virgil
authors.initials: V
authors.lastname: Helaine
authors.forename: Laurence
authors.initials: L
authors.lastname: Hecquet
authors.forename: Wolf-Dieter
authors.initials: WD
authors.lastname: Fessner
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010637:Phenolsulfonphthalein / Q000032:analysis; D011773:Pyruvates / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014174:Transketolase / Q000378:metabolism*
chemical_list: D011773:Pyruvates; C012375:hydroxypyruvic acid; D014174:Transketolase; D010637:Phenolsulfonphthalein