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

Abstract

For the .alpha. 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 is due to the interaction between indole bound to the effector site and the active center of the monomeric .alpha. 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 are thus excluded. A quantitative fit of the data was obtained to an ordered addition mechanism with D-glyceraldehyde 3-phosphate binding first and with a distribution of the enzyme between 2 states differing in V [velocity], governed by the binding of indole to the effector site. The kinetic constants obtained for the .alpha. subunit were compared with those of the .alpha.2.beta.2 complex of tryptophan synthase. Protein-protein interaction of the .alpha. subunit with the .beta.2 subunit does not alter the catalytic mechanism of indoleglycerol phosphate synthesis; suppresses the substrate activation by indole: and changes the various equilibrium, rate and steady-state constants in the sense of conveying higher substrate specificity and catalytic efficiency to the .alpha.-subunit. The occurrence of local and gross conformational changes in the tryptophan synthase system is discussed.