Functional role of cysteinyl residues in tryptophanase.
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 microM for pyridoxal 5'-phosphate with Km of 2.0 microM 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.
D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D003545:Cysteine / Q000032:analysis / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D010084:Oxidation-Reduction; D013048:Specimen Handling; D013861:Thiocyanates / Q000494:pharmacology; D014368:Tryptophanase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D013861:Thiocyanates; D008190:Lyases; D014368:Tryptophanase; D003545:Cysteine; C008658:2-nitro-5-thiocyanobenzoic acid; D004229:Dithiothreitol