Substrate interactions with the alpha-subunit of the Escherichia coli tryptophan synthase. a kinetic study of the wild-type alpha-subunit
Hodo, H.G.; Murphy, J.; Hardman, J.K.; Myers, R.
Archives of Biochemistry and Biophysics 181(2): 419-427
ISSN/ISBN: 0003-9861 PMID: 332076 DOI: 10.1016/0003-9861(77)90247-8
The kinetics of the reversible reaction [indoleglycerol phosphate (In GP) ⇌ indole and glyceraldehyde 3-phosphate] catalyzed by the α-subunit of the Escherichia coli tryptophan synthase was examined. The forward reaction (In Gp to indole) is characterized by simple Michaelis-Menton kinetics at low ( 2 mm) In Gp concentrations, there is a very sharp decrease in rate. This concentration-dependent rate decrease parallels a sharp concentration-dependent aggregation of In GP. This rate decrease at high In Gp concentrations may be due partially to this aggregation and the consequent lowering of monomeric In Gp concentration. The reverse reaction (indole to In GP) is characterized by a sigmoidal response of rate of indole concentration. The data (Hill plot and double reciprocal 1v 1 v versus 1/[indole]2 plot) are consistent with two strongly interacting sites, an effector site and the active site. The effect of In Gp on the reverse reaction is In Gp concentration dependent. At low In Gp 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 In Gp binding only to the glyceraldehyde 3-phosphate portion of the active site. At high In Gp 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 (1v 1 v versus 1/[indole]2) is linear only at high indole concentrations. These data are consistent with the interpretation that In Gp (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 mm indole on the forward reaction is negligible or slightly stimulatory. At low In Gp concentrations, the effect of indole may be due to a diminished activation of the active site when it is occupied by In Gp rather than when it is occupied by indole and glyceraldehyde 3-phosphate. At high In Gp concentrations, the effect of indole can be attributed to the displacement of In Gp by indole in the In Gp aggregate.