Functional role of cysteinyl residues in tryptophanase

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Abstract
Holotryptophanase inactivated by oxidation of cysteinyl residues showed a different absorption spectrum from the native enzyme. At pH 8.0, the native enzyme preferentially existed as a 337‐nm species (active form), whereas in the inactive enzyme a 420‐nm species (inactive form) was dominant. During the reactivation of the enzyme by reduction with dithiothreitol, an increase at 337 nm and a decrease at 420 nm were observed with concomitant increase in enzymatic activity, which was accompanied by the appearance of two cysteinyl residues per monomer. Specific S‐cyanylation of cysteinyl residues by nitrothiocyanobenzoic‐acid‐inactivated apotryptophanase with the modification of one cysteinyl residue per monomer, whereas holotryptophanase was highly resistant to inactivation with nitrothiocyanobenzoic acid. The essential role of the active‐site‐bound pyridoxal 5′‐phosphate in protection against inactivation was confirmed by the agreement of the K1/2 (protection) of 5.0 μM for pyridoxal 5′‐phosphate with Km of‐ 2.0 μM in enzyme catalysis. The inactivation by nitrothiocyanobenzoic acid caused a similar shift in the equilibrium between the 337‐nm species and 420‐nm species, i.e. decrease of the 337‐nm species and increase of the 420‐nm species. From the pH dependence of the equilibrium between these two species, pKa of 7.9 and 7.4 was obtained for the inactive and the dithiothreitol‐activated enzyme, respectively, indicating that cysteinyl residue(s) participated in lowering the pKa of the interconversion between the 337‐nm species (active form) and 420‐nm species (inactive form). The possible role of cysteinyl residues in the function of tryptophanase is discussed.