Assignment of ozone-sensitive tryptophan residue in tryptophanase by a dual-monitoring high-performance liquid chromatography system.
Tryptophanase purified from Escherichia coli B/1t7-A is inactivated by mild ozonization following pseudo-first-order kinetics. Previous data from the authors suggest that one out of two tryptophan residues (Trp's) in the enzyme subunit is preferentially oxidized concomitant with the ozone inactivation and has a direct interaction with the coenzyme, pyridoxal phosphate [PLP (M. Tokushige, Y. Fukuda, and Y. Watanabe, 1979, Biochem. Biophys. Res. Commun. 86, 976-981)]. To determine which Trp is more susceptible to ozonization and interacts with PLP, the native and ozonized enzyme proteins were cleaved by trypsin and the two Trp-containing peptides were analyzed by reverse-phase HPLC equipped with a dual-monitoring system consisting of an uv and a fluorescence monitor connected in tandem for selective detection of Trp-containing peptides. This device facilitated rapid detection and quantitation of the Trp-containing peptides which decreased upon ozonization. The results showed that Trp preferentially oxidized upon ozonization and involved in the interaction with PLP was the one in peptide T-15 rather than that in T-23, which Kagamiyama et al. originally designated (H. Kagamiyama, H. Wada, H. Matsubara, and E. E. Snell, 1972, J. Biol. Chem. 247, 1576-1585).
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors*; D010126:Ozone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D014364:Tryptophan; D014368:Tryptophanase / Q000037:antagonists & inhibitors* / Q000378:metabolism
D010446:Peptide Fragments; D010126:Ozone; D014364:Tryptophan; D008190:Lyases; D014368:Tryptophanase