title: Isolation and properties of human transketolase.
abstract: Recombinant human (His)(6)-transketolase (hTK) was obtained in preparative amounts by heterologous expression of the gene encoding human transketolase in Escherichia coli cells. The enzyme, isolated in the form of a holoenzyme, was homogeneous by SDS-PAGE; a method for obtaining the apoenzyme was also developed. The amount of active transketolase in the isolated protein preparation was correlated with the content of thiamine diphosphate (ThDP) determined in the same preparation. Induced optical activity, facilitating studies of ThDP binding by the apoenzyme and measurement of the transketolase reaction at each stage, was detected by circular dichroism spectroscopy. A single-substrate reaction was characterized, catalyzed by hTK in the presence of the donor substrate and in the absence of the acceptor substrate. The values of the Michaelis constant were determined for ThDP and a pair of physiological substrates of the enzyme (xylulose 5-phosphate and ribose 5-phosphate).
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia. luda@genebee.msu.ru
authors.forename: L E
authors.initials: LE
authors.lastname: Meshalkina
authors.forename: O N
authors.initials: ON
authors.lastname: Solovjeva
authors.forename: Yu A
authors.initials: YA
authors.lastname: Khodak
authors.forename: V L
authors.initials: VL
authors.lastname: Drutsa
authors.forename: G A
authors.initials: GA
authors.lastname: Kochetov
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D010428:Pentosephosphates / Q000737:chemistry / Q000378:metabolism; D012267:Ribosemonophosphates / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014174:Transketolase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D010428:Pentosephosphates; D012267:Ribosemonophosphates; C031626:ribose-5-phosphate; C031625:xylulose-5-phosphate; D014174:Transketolase