N-(5-PHOSPHORIBOSYL)ANTHRANILATE ISOMERASE-INDOLEGLYCEROL-PHOSPHATE SYNTHASE .1. SUBSTRATE-ANALOG BINDS TO 2 DIFFERENT BINDING-SITES ON THE BIFUNCTIONAL ENZYME FROM ESCHERICHIA-COLI

The binding of a fluorescent substrate analog to a bifunctional enzyme of the tryptophan biosynthesis pathway was studied at equilibrium by equilibrium dialysis, steady-state kinetics, fluorescence titration and difference spectroscopy. The substrate analog was obtained by reducing 1-(2-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) with sodium borohydride. The reduced CdRP (rCdRP) was identified with N-(5-phosphoribityl)anthranilate. Equilibrium dialysis in 0.1 M Tris buffer, pH 7.5, 20.degree. C, gives 2 different binding sites for rCdRP on the monomeric, bifunctional PRA [N-(5-phosphoribosyl)anthranilate] isomerase-InGP [indoleglycerol-phosphate] synthase from E. coli. Kd,1 equals 0.2 .mu.M; Kd,2 equals 12.5 .mu.M. These measurements agree with the values of the Kd obtained from fast-reaction kinetics. Steady-state inhibition constants show that the high-affinity binding site is identical with the active site of InGP synthase. This activity is also competitively inhibited by indolepropanol phosphate (IPP), a product analog of InGP. The absorption and fluorescence spectra of rCdRP change upon binding to each of the 2 different binding sites. Fluorometric titrations confirm the value of the dissociation constant of the tight enzyme-ligand complex. Upon replacement of Tris buffer with 0.1 M phosphate buffer, pH 7.6, 20.degree. C, the values of Km for CdRP and Ki [inhibition constant] for rCdRP, InGP and IPP increase approximately 40-fold, whereas the dissociation constant of the loose enzyme-ligand complex is affected only moderately.