N-(5-Phosphoribosyl)anthranilate isomerase-indoleglycerol-phosphate synthase. 2. Fast-reaction studies show that a fluorescent substrate analogue binds independently to two different sites.
The mechanism of binding of reduced 1-(2-carboxyphenylamino)-1-deoxyribulose 5-phosphate (rCdRP) to two different binding sites on the bifunctional enzyme is determined by kinetic studies, using temperature-jump and stopped-flow equipment with fluorescence detection. Two rapid binding processes and a comparatively slow isomerization process are observed over a wide range of enzyme and rCdRP concentrations. Kinetic measurements with low concentrations of rCdRP show that the isomerization is coupled only to the more rapid of the two binding reactions that involves the active site of indoleglycerol-phosphate synthase. The slower of the two binding reactions represents rCdRP binding in one step to the active site of (phosphoribosyl)anthranilate isomerase. The simplest mechanism explaining quantitatively the dependence of the relaxation times on concentration consists of rCdRP binding to two sites on the enzyme that are intrinsically different and independent, even to the extent that a ligand-induced isomerization of one site is not transmitted to the other site. Simulation studies show that the concentration dependences of the amplitudes of the three relaxation processes are also consistent with the mechanism. The results are discussed in terms of two autonomous domains of folding of the polypeptide chain.
D002262:Carboxy-Lyases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000378:metabolism*; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009097:Multienzyme Complexes / Q000378:metabolism*; D012267:Ribosemonophosphates; D012274:Ribulosephosphates; D013050:Spectrometry, Fluorescence; D013696:Temperature; D062367:ortho-Aminobenzoates
D009097:Multienzyme Complexes; D012267:Ribosemonophosphates; D012274:Ribulosephosphates; D062367:ortho-Aminobenzoates; D002262:Carboxy-Lyases; D007209:Indole-3-Glycerol-Phosphate Synthase; D007535:Isomerases