Modification of tryptophanase 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-inactivation. The essential importance of the active-site-bound pyridoxal 5'-phosphate (pyridoxal-P) for the protection was confirmed by the agreement of K 1/2 (protection) (1.2 microM) for pyridoxal-P with Km (1.5 microM) in enzyme catalysis. Amino acid analyses and inactivation stoichiometry showed that modification of 1--2 tyrosyl residues per monomer caused complete inactivation. The appearance of 430-nm species upon incubation of C(NO2)4-inactivated apoenzyme with pyridoxal-P indicated that the C(NO2)4-inactivated apoenzyme could still bind the coenzyme, although an affinity of the enzyme for pyridoxal-P (Kd = 51 microM) was much lower than that of the native enzyme (Kd = 0.7 microM). A close relationship was observed between the cofactor activity of monovalent cations and their effectiveness in the protection by pyridoxal-P: in the presence of active monovalent cations (K+, NH+4 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 monovalent cations. From the experimental results obtained it was suggested strongly that tryptophanase has essential tyrosyl residues near the active site. The tyrosyl residues were prevented from the attack of C(NO2)4 by the active-site-bound pyridoxal-P only in the catalytically active holoenzyme.
D000596:Amino Acids / Q000032:analysis; D002414:Cations, Monovalent; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D008697:Methane / Q000031:analogs & derivatives*; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013774:Tetranitromethane / Q000494:pharmacology*; D014368:Tryptophanase / Q000378:metabolism*
D000596:Amino Acids; D002414:Cations, Monovalent; D011732:Pyridoxal Phosphate; D008190:Lyases; D014368:Tryptophanase; D013774:Tetranitromethane; D008697:Methane