Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 220.127.116.11) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo.
Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine
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G D Smith, D V Roberts, A Daday; Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine. Biochem J 1 July 1977; 165 (1): 121–126. doi: https://doi.org/10.1042/bj1650121
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