Substrate interactions with the α-subunit of the Escherichia coli tryptophan synthase : A kinetic study of the wild-type α-subunit

Abstract

The kinetics of the reversible reaction [indoleglycerol phosphate (InGP) ⇌ indole and glyceraldehyde 3-phosphate] catalyzed by the α-subunit of the Escherichia coli tryptophan synthase was examined. The forward reaction (InGP to indole) is characterized by simple Michaelis-Menton kinetics at low (<2 m m) InGP concentrations and is consistent with the binding of a single InGP molecule at the active site. At higher (>2 m m) InGP concentrations, there is a very sharp decrease in rate. This concentration-dependent rate decrease parallels a sharp concentration-dependent aggregation of InGP. This rate decrease at high InGP concentrations may be due partially to this aggregation and the consequent lowering of monomeric InGP concentration. The reverse reaction (indole to InGP) is characterized by a sigmoidal response of rate of indole concentration. The data (Hill plot and double reciprocal 1 v versus 1/[indole] 2 plot) are consistent with two strongly interacting sites, an effector site and the active site. The effect of InGP on the reverse reaction is InGP concentration dependent. At low InGP concentrations, there is an inhibition with a concomitant slight decrease in the V. Under these conditions, the two indole sites are still apparent. These data are interpreted as being due to monomeric InGP binding only to the glyceraldehyde 3-phosphate portion of the active site. At high InGP concentrations, there is an increased activation of the reverse reaction and nearly a doubling in V. In addition, the Hill slope approaches a value of 2 and the double-reciprocal plot ( 1 v versus 1/[indole] 2) is linear only at high indole concentrations. These data are consistent with the interpretation that InGP (probably the aggregate form) binds to a separate effector site and that this binding precludes the functioning of the indole effector site at low indole concentrations. The effect of 1 or 7 m m indole on the forward reaction is negligible or slightly stimulatory. At low InGP concentrations, the effect of indole may be due to a diminished activation of the active site when it is occupied by InGP rather than when it is occupied by indole and glyceraldehyde 3-phosphate. At high InGP concentrations, the effect of indole can be attributed to the displacement of InGP by indole in the InGP aggregate.