Structural and Functional Roles of the Cysteine Residues in the α Subunit of the Escherichia coli Tryptophan Synthetase

Abstract

Abstract The reaction pattern of the three sulfhydryl groups of the α subunit of the Escherichia coli tryptophan synthetase with N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) at pH 7.0 is profoundly altered in the presence of 10 mm indole. The effect of indole is upon both the rate of reaction, and upon its over-all extent. In the absence of indole, only cysteinyl sulfhydryl groups 80 and 117 of the α subunit are available for reaction with N-ethylmaleimide, but in 10 mm indole all three thiol groups become available for reaction. Similar results were obtained with DTNB (i.e. in the absence of indole a single sulfhydryl group reacts rapidly with DTNB, any further reaction occurring very slowly; in the presence of indole all three sulfhydryl groups react at a slower although uniform rate). The effects of indole in the 7.5 to 10.0 mm concentration range could not be mimicked with 20% sucrose, 20% ethanol, 20% ethylene glycol, or 20% dioxane, solvents known to be effective perturbants of protein tertiary structure. The presence of indole also had no effect on the optical rotatory dispersion of the enzyme, and caused no detectable change in the 16 ± 2% α helical structure found for this protein. Indole 3-glycerol phosphate (InGP) at concentrations twice the enzyme's Km for this substrate in the InGP aldolase reaction catalyzed by the free α subunit, protected all three sulfhydryl groups from reaction with DTNB in the presence of indole. Examination of the indole concentration dependence of its effect on sulfhydryl group reactivity, and determination of its Km in the reverse InGP aldolase reaction (synthesis of InGP from indole in the presence of excess d-glyceraldehyde 3-phosphate) showed that the two processes reached half-saturation at indole concentrations of 3.6 and 4.1 mm, respectively. It is concluded that the effect of indole on sulfhydryl group reactivity reflects enzyme conformational changes related to the mechanism of catalysis of the InGP aldolase reaction by the independent α subunit. Indole-induced conformational changes in α subunit structure may be involved in the regulation and catalysis of the last steps in tryptophan synthesis by the α2β2-tryptophan synthetase complex.