Transketolase a of Escherichia coli K12

• DOI: 10.1111/j.1432-1033.1995.0525h.x
• Publication Date: 2005-03-04T12:49:44Z
• Authors: Georg A. Sprenger; Ulrich Schörken; Gerda Sprenger; Hermann Sahm
• Keywords: Transketolase

Transketolase A was purified to apparent homogeneity from recombinant Escherichia coli K12 cells carrying the homologous cloned tktA gene on a pUC19‐derived plasmid. These exhibited transketolase activity in crude extracts of up 9.7 U/mg compared ≤0.1 wild‐type cells. strain by successive ammonium sulfate precipitations and two anion‐exchange chromatography steps (Q‐Sepharose FF, Fractogel EMD‐DEAE column) afforded an apparently homogeneous protein band SDS/PAGE. The enzyme, both its active apoform, had molecular mass 145000 Da (±10000 Da), judged gel‐filtration chromatography. Subunits 73000 (±2000 Da) were determined SDS/PAGE, thus, most likely forms homodimer. N‐terminal amino acid sequencing verified identity with tktA. specific at 30°C substrates xylulose 5‐phosphate (donor C2 compound) ribose (acceptor) optimal pH (50 mM glycylglycine, 8.5), 50.4 U/mg. K m values for 160μM 1.4mM, respectively. other physiological 90 μM erythrose 4‐phosphate (best acceptor substrate), 2.1 D,L‐glyceraldehyde 3‐phosphate, 1.1 fructose 6‐phosphate, 4 sedoheptulose 7‐phos‐phate. Hydroxypyruvate served as alternative donor ( =18 mM). Unphosphorylated compounds formaldehyde = 31 mM), glycolaldehyde (14 (10 mM) d ‐erythrose (150 enzyme competitively inhibited ‐arabinose i 6 concentration 2.5 5‐phosphate) or chelating agent EDTA. inactive apoform yielded dialysis against buffer containing 10 EDTA, thus removing cofactors thiamine diphosphate divalent cations. reconstitution apoenzyme pro‐ceded faster presence manganese ions 7 diphosphate) than