Modification of Trytophanase with Tetranitromethane

Modification of apotryptophanase with tetranitromethane [C(NO2)4] resulted in a loss of enzymatic activity, whereas holotryptophanase was highly resistant against C(NO2)4‐inativation. The essential importance of the active‐site‐bound pyridoxal 5′‐phosphate (pyridoxalP) for the protection was confirmed by the agreement of K1/2 (protection) (1.2 μM) for pyridoxal‐P with Km (1.5μM) in enzyme catalysis. Amino acid analyses and inactivation stoichimetry showed that modification of 1‐2 tyrosyl residues per monomer caused completee inactivation. The appearance of 430‐nm species upon incubation of C(NO2)4‐inactivated apoenzyme with pyridoxal‐P indicated that the (NO2)4‐inactivated apoenzyme could still bind the coenzyme, although an affinity of the enzyme for pyridoxal‐P(Kd= 51μM) was much lower than that of the native enzyme (Kd= 0.7μM). A close relationship was observed between the cofactor activity of monovalent cations and their effecctiveness in the protection yb pyridoxal‐P: in the presence of active monovalent cations (K+, NH4+ and Rb+) pyridoxal‐P could provide the protection but not in the presence of inactive cations (Li+, Na+ and Cs+) as well as in the absence of inorganic nonov