18O studies of pyrogallol cleavage by catechol 1,2-dioxygenase.
18O labeling studies on the catechol 1,2-dioxygenase-catalyzed oxidative cleavage of pyrogallol demonstrate that the enzyme functions both as a dioxygenase and a monooxygenase in this reaction. Two products are observed, 2-pyrone-6-carboxylic acid, 99% singly labeled at the carboxylate, and 2-hydroxy-cis,cis-muconic acid, 74% doubly labeled (one 18O at each carboxylate) and 24% single labeled (one 18O at either carboxylate). The labeling pattern observed shows that 2-pyrone-6-carboxylic acid cannot be derived enzymatically from the lactonization of the 2-hydroxy-cis,cis-muconic acid, thus eliminating the dioxetane as an intermediate in the dioxygenase mechanism. The observations are interpreted to indicate the intermediacy of 2-hydroxymuconic anhydride. This anhydride or the corresponding muconyl enzyme species must be sufficiently long-lived to allow the exchange of labeled hydroxide with solvent. Evidence for mechanism-based enzyme inactivation by a pyrogallol-derived intermediate is also presented.
D050548:Catechol 1,2-Dioxygenase; D049308:Dioxygenases; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D010103:Oxygen Isotopes; D010105:Oxygenases / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology; D011748:Pyrogallol / Q000378:metabolism*
D010103:Oxygen Isotopes; D011748:Pyrogallol; D010105:Oxygenases; D049308:Dioxygenases; D050548:Catechol 1,2-Dioxygenase