Purification and characterization of anthranilate synthase from Catharanthus roseus.
Anthranilate synthase (EC 4.1.3.27) has been purified from cell cultures of Catharanthus roseus by poly(ethylene glycol) precipitation/fractionation and subsequent separation by anion exchange on Q-Sepharose, Orange A dye chromatography, Mono Q anion-exchange chromatography and Superose 6 gel filtration. By analogy to anthranilate synthases from other sources it does look like the enzyme is a tetramer composed of two large and two small subunits, with molecular mass 67 and 25.5 +/- 0.5 kDa, respectively. The molecular mass determined by gel filtration was 143 +/- 5 kDa. The enzyme had a pI of 5.1 determined by chromatofocusing. The pH optimum was between pH 7.5 and pH 8.3, but the type of buffer used affected the results. The enzyme could utilize NH4+ as ammonium donor instead of glutamine. The enzyme showed normal Michaelis-Menten kinetics with respect to the substrates L-glutamine and chorismate, and the cofactor Mg2+, Km values for L-glutamine was determined to be 0.37 +/- 0.05 mM, for chorismate 67 +/- 3 microM, and for MgCl2 0.26 +/- 0.03 mM respectively. Anthranilate synthase was inhibited by L-tryptophan, tryptamine and D-tryptophan (with L-tryptophan being the best inhibitor). The enzyme was allosterically regulated showing positive cooperatively of chorismate binding at higher concentrations of tryptophan. For a tryptophan concentration of 20 microM the Hill coefficient was determined to be 2. The tryptophan binding sites showed positive cooperatively for higher concentrations of chorismate. The purified enzyme did not contain anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity and is thus not of the same type as the well characterized Salmonella typhimurium anthranilate synthase/phosphoribosyl pyrophosphate transferase bifunctional type.
Poulsen
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C
Center for Bio-Pharmaceutical Sciences, University of Leiden, The Netherlands.
Bongaerts
R J
RJ
Verpoorte
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D000878:Anthranilate Synthase / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D001419:Bacteria / Q000201:enzymology; D002478:Cells, Cultured; D004795:Enzyme Stability; D005246:Feedback; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000032:analysis; D007700:Kinetics; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity; D014364:Tryptophan / Q000494:pharmacology
D007527:Isoenzymes; D014364:Tryptophan; D000878:Anthranilate Synthase