TRANSKETOLASE-A OF ESCHERICHIA-COLI K12 - PURIFICATION AND PROPERTIESOF THE ENZYME FROM RECOMBINANT STRAINS

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 recombinant cells exhibited a transket olase activity in crude extracts of up to 9.7 U/mg compared to less th an or equal to 0.1 U/mg in wild-type cells. Transketolase A was purifi ed from crude extracts of a recombinant strain by successive ammonium sulfate precipitations and two anion-exchange chromatography steps (Q- Sepharose FF, Fractogel EMD-DEAE column) and afforded an apparently ho mogeneous protein band on SDS/PAGE. The enzyme, both in its active and apoform, had a molecular mass of 145 000 Da (+/- 10000 Da), judged by gel-filtration chromatography. Subunits of 73 000 Da (+/- 2000 Da) we re determined on SDS/PAGE, thus, transketolase A most likely forms a h omodimer. N-terminal amino acid sequencing of the protein verified the identity with the cloned gene tktA. The specific activity of the puri fied enzyme, determined at 30 degrees C with the substrates xylulose 5 -phosphate (donor of C2 compound) and ribose 5-phosphate (acceptor) at an optimal pH (50 mM glycylglycine, pH 8.5), was 50.4 U/mg. K-m value s for the substrates xylulose 5-phosphate and ribose 5-phosphate were 160 mu M and 1.4 mM, respectively. K,values for the other physiologica l substrates of transketolase A were 90 mu M for erythrose 4-phosphate (best acceptor substrate), 2.1 mM for D,L-glyceraldehyde 3-phosphate, 1.1 mM for fructose 6-phosphate, and 4 mM for sedoheptulose 7-phospha te. Hydroxypyruvate served as alternative donor (K-m = 18 mM). Unphosp horylated acceptor compounds were formaldehyde (K-m = 31 mM), glycolal dehyde (14 mM), D,L-glyceraldehyde (10 mM) and D-erythrose (150 mM). T he enzyme was competitively inhibited by D-arabinose 5-phosphate (K-i = 6 mM at a concentration of 2.5 mM D-arabinose 5-phosphate) or by the chelating agent EDTA. The inactive apoform of transketolase A was yie lded by dialysis against buffer containing 10 mM EDTA, thus removing t he cofactors thiamine diphosphate and divalent cations. The reconstitu tion of the apoenzyme proceded faster in the presence of manganese ion s (K-d = 7 mu M at 10 mu M thiamine diphosphate) than with other dival ent cations.