46] Chorismate to tryptophan (Escherichia coli)—anthranilate synthetase, PR transferase, PRA isomerase, InGP synthetase, tryptophan synthetase

Abstract

This chapter describes the characteristics of the enzymes and intermediates involved in the biosynthesis of tryptophan from chorismate in microorganisms. N-5′-phosphoribosyl anthranilate (PRA) is an unstable intermediate, which may be produced synthetically by mixing at room temperature equal volumes of 1.0 M ribose-5-P in water and 1.0 M anthranilic acid dissolved in 95% ethanol. The synthesis of indole-3-glycerol phosphate (InGP) involves enzymatic synthesis from anthranilic acid and ribose 5-phosphate via PRA and 1-( o -carboxyphenylamino)-l-deoxyribulose 5-phosphate (CdRP). InGP is quite stable and may be kept for months at -15º. In context to enzyme assays, anthranilate synthetase activity is most conveniently measured by following anthranilate formation fluorometrically. The presence of PR transferase in the enzyme preparation generally does not present any difficulties, because anthranilate utilization is negligible in the absence of phosphoribosyl pyrophosphate (PRPP). PRA isomerase activity is best assayed by following InGP formation spectrophotometrically in the presence of excess InGP synthetase.