Active site-directed modification of tryptophanase by 3-bromopyruvate.
Tryptophanase purified from Escherichia coli B/It7-A was irreversibly inactivated by 3-bromopyruvate following pseudo-first-order kinetics. The inactivation rate for the holoenzyme tended to saturate as the concentration f bromopyruvate increased. L-Alanine and DL-3-phenylserine, potent competitive inhibitors with respect to L-tryptophan decomposition, protected the enzyme from inactivation. Titration of SH groups in the enzyme protein with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) showed that modification of one SH group per enzyme subunit resulted in a complete inactivation. When the enzyme was subjected to bromopyruvate-modification following pretreatment with DTNB, the activity was almost completely restored upon reduction with dithiothreitol. Modification of the enzyme with bromopyruvate quenched the absorption peak near 500 nm, characteristic of a quinoidal structure formed by labilization of the alpha-proton. These results support the possibility that bromopyruvate reacts with the enzyme as an affinity labeling agent.
D000345:Affinity Labels; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D004228:Dithionitrobenzoic Acid; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors*; D011773:Pyruvates / Q000494:pharmacology*; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000032:analysis; D013696:Temperature; D014368:Tryptophanase / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000345:Affinity Labels; D011773:Pyruvates; D013438:Sulfhydryl Compounds; C017092:bromopyruvate; D004228:Dithionitrobenzoic Acid; D008190:Lyases; D014368:Tryptophanase