Abstract
Properties and regulation of anthranilate synthase from Alcaligenes eutrophus H 16 were investigated. Anthranilate synthase was partially purified from crude extracts by affinity chromatography on tryptophan-substituted Sepharose, and was used for kinetic measurements. During the purification procedure the enzyme was stabilized by 50 mM l-glutamine or during chromatography on DEAE-cellulose and Sephadex G-200 with 30% glycerol, respectively.
The glutamine dependent activity of anthranilate synthase was examined; it showed little change between pH 8.4 and pH 9.1. The Arrhenius plot was broken and the activation energy, δH, calculated therefrom amounted to 8.9 kcal/mole up to 30°C and 5.5 kcal/mole at higher temperatures. The molecular weight determined by gelfiltration on Sephadex G-200 and by sucrose density gradient centrifugation resulted in 158000 and 126000, respectively. The K m -values for the two substrates chorismate and glutamine were found to be 5 μM and 560 μM, respectively.
Anthranilate synthase was strongly inhibited by l-tryptophan; the only amino acid that affected enzyme activity. Homotropic interactions for chorismate (Hill coefficient n=1.4) were obtained in the presence of l-tryptophan. 50% inhibition were caused by 10 μM l-tryptophan at 100 μM chorismate. The inhibition with respect to l-glutamine was noncompetitive.
Anthranilate synthase was not associated to phosphoribosyl transferase and easily separable from the latter by different chromatographic methods.
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Abbreviations
- TEA:
-
triethanolamine
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Friedrich, C.G., Friedrich, B. & Schlegel, H.G. Aromatic amino acid biosynthesis in Alcaligenes eutrophus H 16 . Arch. Microbiol. 107, 125–131 (1976). https://doi.org/10.1007/BF00446831
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DOI: https://doi.org/10.1007/BF00446831