Open AccessThe Mechanism of the Synthesis of Indoleglycerol Phosphate Catalyzed by Tryptophan Synthase from Escherichia coli
Author(s) -
WEISCHET Wolf gang O.,
KIRSCHNER Kasper
Publication year - 1976
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1976.tb10350.x
Subject(s) - phosphate , indole test , product inhibition , tryptophan synthase , chemistry , kinetics , atp synthase , steady state (chemistry) , stereochemistry , tryptophan , substrate (aquarium) , escherichia coli , enzyme , catalysis , non competitive inhibition , biochemistry , organic chemistry , biology , amino acid , ecology , physics , quantum mechanics , gene
The mechanism of indoleglycerol phosphate synthesis from indole and d ‐glyceraldehyde 3‐phosphate catalyzed by tryptophan synthase has been investigated by steady‐state kinetic techniques. The equilibrium constant and the progress curves were measured by use of the difference in absorbance between indole and indoleglycerol phosphate. Stopped‐flow measurements show that only the nonhydrated form of d ‐glyceraldehyde 3‐phosphate serves as substrate. The product analogue indolepropanol phosphate was used as an inhibitor to discriminate between possible mechanisms. The data agree well with an ordered addition mechanism with d ‐glyceraldehyde 3‐phosphate adding first. Mechanisms involving random addition of substrates or ordered addition with indole adding first can he excluded because indolepropanol phosphate is a competitive inhibitor only towards glyceraldehyde 3‐phosphate. The high affinity of tryptophan synthase for indoleglycerol phosphate leads to product inhibition even at small extents of reaction. Glyceraldehyde 3‐phosphate combines with the enzyme with an apparent second‐order rate constant, which is not diffusion controlled and generates a site with. high affinity for indole