Chemical modification of tryptophanase by chloramine T: a possible involvement of the methionine residue in enzyme activity.
Tryptophanase purified from Escherichia coli B/1t7-A was irreversibly inactivated by chloramine T (sodium N-chloro-p-toluenesulfonamide). The mode of inactivation was rather complex and did not follow pseudo-first-order kinetics. The inactivation of the apoenzyme was much faster than that of the holoenzyme. The Km value for the synthetic substrate S-o-nitrophenyl-L-cysteine (SOPC) increased concomitantly with the modification. In contrast, the Km value for the coenzyme, pyridoxal 5'-phosphate (PLP), was not altered. L-Serine, another substrate, and L-alanine, a competitive inhibitor, protected the enzyme from inactivation. Determination of SH groups in the enzyme protein with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) showed that modification of two SH groups per enzyme subunit resulted in a complete inactivation. When the enzyme was subjected to chloramine T-modification following the SH group modification with DTNB, further inactivation was still observed, even after the addition of dithiothreitol. The SH-blocked enzyme preparation thus obtained, however, exhibited less pH dependency of inactivation by chloramine T than that of the native enzyme. The amino acid analysis of the chloramine T-modified enzyme showed that modification of four or five methionine residues among the 16 residues per subunit proceeded concomitantly with the complete inactivation. Modification of the enzyme with chloramine T quenched the absorption peak near 500 nm, characteristic of a quinoidal structure formed by labilization of the alpha-proton. These results suggest the possibility that chloramine T modifies not only the SH groups, but also methionine residues important for the catalytic activity of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
D000596:Amino Acids / Q000032:analysis; D000891:Anti-Infective Agents, Local / Q000633:toxicity*; D002700:Chloramines / Q000633:toxicity*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008190:Lyases / Q000378:metabolism; D008715:Methionine; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014105:Tosyl Compounds; D014368:Tryptophanase / Q000378:metabolism
D000596:Amino Acids; D000891:Anti-Infective Agents, Local; D002700:Chloramines; D013439:Sulfhydryl Reagents; D014105:Tosyl Compounds; C016300:chloramine-T; D008715:Methionine; D008190:Lyases; D014368:Tryptophanase