Indole Channeling by Tryptophan Synthase of Neurospora

• Published in: The Journal of biological chemistry Vol. 249; no. 13; pp. 4041 - 4049
• Main Author: Matchett, William H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.07.1974; American Society for Biochemistry and Molecular Biology
• Subjects: Binding Sites; Biological Evolution; Carbon Radioisotopes; Glycerophosphates - metabolism; Indoles - metabolism; Kinetics; Mathematics; Neurospora - enzymology; Neurospora crassa - enzymology; plant biochemistry; plant physiology; Pyridoxal Phosphate - pharmacology; Serine - pharmacology; Time Factors; Tryptophan - biosynthesis; Tryptophan Synthase - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)42481-2

Tryptophan synthase (l-serine hydro-lyase-adding indole) (EC 4.2.1.20) of Neurospora crassa was studied in reaction mixtures containing [2-14C]indoleglycerolphosphate and non-labeled indole. A detailed kinetic analysis of the passage of isotope from indoleglycerolphosphate to indole and to tryptophan during the synthesis of tryptophan revealed that: (a) indole disappears first and indoleglycerolphosphate is utilized after the indole concentration falls to barely detectable levels; (b) significant amounts of radioactivity appear in indole; and (c) the specific radioactivity of the tryptophan formed is much lower than that of indoleglycerolphosphate but 2- to 5-fold higher than that of indole depending inversely upon the initial indole concentration. The analyses of these observations are interpreted as evidence that indole is an enzyme-bound or “channeled” intermediate in the conversion of indoleglycerolphosphate to tryptophan. The enzyme was separated from reaction mixtures containing an equilibrium mixture of labeled indoleglycerolphosphate and labeled indole by passage of the mixtures through ultrafilters which retained material of molecular weights greater than 50,000. Radioactive indole was found exchangeably bound to the retained enzyme. From these results, it was estimated that 2 to 3 moles of indole were bound per mole of enzyme. The results are interpreted as evidence that the Neurospora enzyme fits well the conceptual framework of the “surface model” (Davis, R. H. (1967) in Organizational Biosynthesis (Vogel, H. J., Lampen, J. O., and Bryson, V., eds) pp. 303–322, Academic Press, New York) and that the enzyme may have evolved, as first suggested (Bonner, D. M., DeMoss, J. A., and Mills, S. E. (1965) in Evolving Genes and Proteins (Bryson, V., and Vogel, H. J., eds) pp. 305–318, Academic Press, New York), from a primitive multicomponent complex.