Steady‐State Kinetic Studies of the Synthesis of Indoleglycerol Phosphate Catalyzed by the α Subunit of Tryptophan Synthase from Escherichia coli

For the α subunit of tryptophan synthase and at constant concentration of d ‐glyceraldehyde 3‐phosphate the saturation curves with respect to indole concentration are weakly sigmoidal. This phenomenon can be explained by interaction between indole bound to the effector site established previously and the active center of the monomeric α subunit. Kinetic studies of the inhibition of indoleglycerol phosphate synthesis by the analogue indolepropanol phosphate show that the inhibition is competitive with respect to d ‐glyceraldehyde 3‐phosphate and non‐competitive with respect to indole. Mechanisms with random addition of substrates or ordered addition with indole binding first can therefore be excluded. A quantitative fit of the data has been obtained to an ordered addition mechanism with d ‐glyceraldehyde 3‐phosphate binding first and with a distribution of the enzyme between two states differing in V , governed by the binding of indole to the efector site. The kinetic constants obtained for the α subunit have been compared with those of the α 2 β 2 complex of tryptophan synthase. Protein‐protein interaction of the α subunit with the β 2 subunit (a)‐does not alter the catalytic mechanism of indoleglycerol phosphate synthesis, (b) suppresses the substrate activation by indole, and (c) changes the various equilibrium, rate and steady‐state constants in the sense of conveying higher substrate specificity and catalytic efficiency to the α‐subunit. The ocurrence of local and gross conformational changes in the tryptophan synthase system is discussed