The mechanism of the synthesis of indoleglycerol phosphate catalyzed by tryptophan synthase from Escherichia coli. Steady-state kinetic studies.
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 non-hydrated 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 be 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.
D055598:Chemical Phenomena; D002621:Chemistry; D005994:Glycerophosphates / Q000138:chemical synthesis*; D066298:In Vitro Techniques; D007211:Indoles / Q000138:chemical synthesis*; D007700:Kinetics; D010710:Phosphates; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005994:Glycerophosphates; D007211:Indoles; D010710:Phosphates; D014367:Tryptophan Synthase