Details

Autor(en) / Beteiligte

• Mendel, Sharon
• Arndt, Andrew
• Bugg, Timothy D. H

Titel

Acid−Base Catalysis in the Extradiol Catechol Dioxygenase Reaction Mechanism:  Site-Directed Mutagenesis of His-115 and His-179 in Escherichia coli 2,3-Dihydroxyphenylpropionate 1,2-Dioxygenase (MhpB)

Ist Teil von

• Biochemistry (Easton), 2004-10, Vol.43 (42), p.13390-13396

Ort / Verlag

United States: American Chemical Society

Erscheinungsjahr

2004

Quelle

MEDLINE

Beschreibungen/Notizen

• The extradiol catechol dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products. Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid−base catalysis for this reaction. Two conserved histidine residues were identified in the active site of the class III extradiol dioxygenases, positioned within 4−5 Å of the iron(II) cofactor. His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine. Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid−base residues. Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid. Mutant H179Q was catalytically active for the lactone hydrolysis half-reaction, whereas mutant H115Q was inactive, implying a role for His-115 in lactone hydrolysis. A catalytic mechanism involving His-179 and His-115 as acid−base catalytic residues is proposed.