Single-turnover kinetics of homoprotocatechuate 2,3-dioxygenase.
Homoprotocatechuate 2,3-dioxygenase isolated from Brevibacterium fuscum utilizes an active site Fe(II) and O(2) to catalyze proximal extradiol cleavage of the substrate aromatic ring. In contrast to other members of the ring cleaving dioxygenase family, the transient kinetics of the extradiol dioxygenase catalytic cycle have been difficult to study because the iron is nearly colorless and EPR silent. Here, it is shown that the reaction cycle kinetics can be monitored by utilizing the alternative substrate 4-nitrocatechol (4NC), which is also cleaved in the proximal extradiol position. Changes in the optical spectrum of 4NC occurring as a result of ionization, environmental changes, and ring cleavage allow both the substrate binding and product formation phases of the reaction to be studied. It is shown that substrate binding occurs in a four-step process probably involving binding to two ionization states of the enzyme at different rates. Following an initial rapid binding of the monoanionic 4NC in the active site, slower binding to the Fe(II) and conversion to the dianionic form occur. The bound dianionic 4NC reacts rapidly with O(2) in four additional steps, apparently occurring in sequence. On the basis of the optical properties of the intermediates, these steps are hypothesized to be O(2) binding to the iron, isomerization of the resulting complex, ring opening, and product release. The natural substrate appears to form the same intermediates but with much larger rate constants. These are the first transient intermediates to be reported for an extradiol dioxygenase reaction.
Groce
Stephanie L
SL
Department of Biochemistry, Molecular Biology, and Biophysics and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Miller-Rodeberg
Marcia A
MA
Lipscomb
John D
JD
D000693:Anaerobiosis; D001665:Binding Sites; D001951:Brevibacterium / Q000201:enzymology; D002384:Catalysis; D002396:Catechols / Q000737:chemistry / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D002396:Catechols; D049308:Dioxygenases; C020055:3,4-dihydroxyphenylacetate 2,3-dioxygenase; D010100:Oxygen; C001833:4-nitrocatechol