Aromatic ring cleavage by homoprotocatechuate 2,3-dioxygenase: Role of His200 in the kinetics of interconversion of reaction cycle intermediates

Stephanie L. Groce, John D. Lipscomb

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63 Scopus citations

Abstract

Homoprotocatechuate 2,3-dioxygenase (WT 2,3-HPCD) isolated from Brevibacterium fuscum utilizes an active site Fe(II) and O2 to catalyze proximal extradiol cleavage of the aromatic ring of the substrate (HPCA). Here, the conserved active site residue His200 is changed to Gln, Glu, Ala, Asn, and Phe, and the reactions of the mutant enzymes are probed using steady-state and transient kinetic techniques. Each mutant catalyzes ring cleavage of HPCA to yield the normal product. H200Q and H200N retain 30-40% of the WT 2,3-HPCD activity at 24°C, but the other mutants reduce the k cat to less than 9% of normal. The origin of the reduced activity is unlikely to be the substrate binding phase of the catalytic cycle, because the multistep anaerobic binding reaction of the chromophoric substrate 4-nitrocatechol (4NC) is shown to proceed with rate constants similar to those observed for WT 2,3-HPCD. In contrast, the rate constants of several steps in the multistep O2 binding/insertion and product release half of the reaction cycle are substantially slowed, in particular the steps in which activated oxygen attacks the organic substrate and in which product is released. In the case of the H200N mutant, the product of 4NC oxidation is not the usual ring cleavage product, but rather the 4NC quinone. These results suggest that the main role of His200 is in facilitating the steps in the second half of the reaction cycle. The decreased rate constants for the O2 insertion steps in the catalytic cycles of the mutant enzymes allow the oxygen adduct of an extradiol dioxygenase to be detected for the first time.

Original languageEnglish (US)
Pages (from-to)7175-7188
Number of pages14
JournalBiochemistry
Volume44
Issue number19
DOIs
StatePublished - May 17 2005

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