Data from steady state and transient kinetics show that the functional phosphoribosyl anthranilate isomerase domain of the naturally bifunctional enzyme from Escherichia coli has properties similar to those of its artificially excised domain. The naturally monofunctional enzyme from Saccharomyces cerevisiae has significantly higher values of both kcat and kcat/KMPRA. The primary product of a single turnover of phosphoribosylanthranilate is fluorescent, but it slowly isomerizes to the nonfluorescent stable product. The latter is the competent substrate of indoleglycerol phosphate synthase, which catalyzes the subsequent step of tryptophan biosynthesis. The isomerization is characterized by a monoexponential decay independent of phosphoribosyl anthranilate isomerase. Due to a tentative assignment of the fluorescent, primary product and the nonfluorescent, stable product to an enol and a keto compound, respectively, tryptophan biosynthesis appears to be rate-limited by an uncatalyzed enol/keto tautomerization. A formal kinetic mechanism of the reaction catalyzed by phosphoribosyl anthranilate isomerase is proposed that is consistent with the combined enzymic and ligand binding properties of the three variants of phosphoribosyl anthranilate isomerase.