Monovalent cation activation of tryptophanase.

The interaction of monovalent cations with holotryptophanase has been examined by spectral and kinetic methods. Using S-orthonitrophenyl-L-cysteine as a substrate, activation by the following monovalent cations was demonstrated; values of KA (mM, in italics) and Vmax (mumol min-1 mg) aare given in parentheses: Li+ (54 +/- 11.6, 4.3 +/- 0.28), Na+ (40 +/- 0.03, 18) K+ (1.44 +/- 0.06, 41.1 +/- 3.5), Tl+ (0.95 +/- 0.1, 39 +/- 4.4), NH4+ (0.23 +/- 0.01, 57.9 +/- 2.6), Rb+ (3.5 +/- 0.3, 33.5 +/- 1.8), Cs+ (14.6 +/- 2.6, 21 +/- 2.3). It was demonstrated by circular dichroic spectra that the competitive inhibitor, ethionine, interacts with the holoenzyme in the absence of activating monovalent cations, although it does not undergo labilization of the alpha proton. On addition of monovalent cation to the holoenzyme-ethionine complex, a marked increase occurs in absorption of 508 nm resulting from labilization of the alpha proton with formation of the quinoid form of the pyridoxal phosphate moiety of the enzyme-substrate complex at the catalytic center (Morino, Y., and Snell, E.E. (1967) J. Biol. Chem; 242, 2800-2809. The extent of formation of this quinoid intermediate was linearly related to the maximum velocity observed with each cation except NH4+, which was anomalously active. When measured at 500 nm, the change in absorption ranged from deltaA = 0.45 mg-1 of tryptophanase for NH4+ to 0.06 mg-1 for Li+. Two moles of thallium (I) were bound per mole of subunit. The data are most consistent with the interaction of monovalent cation at or near the catalytic center in such a way that it either participates directly in the reaction or is required for the critical alignment of one or more functional groups necessary for catalysis.