PMID:15491145
| Citation |
Mendel, S, Arndt, A and Bugg, TD (2004) 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). Biochemistry 43:13390-6 |
|---|---|
| Abstract |
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 A 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. |
| Links |
PubMed Online version:10.1021/bi048518t |
| Keywords |
Binding Sites/genetics; Catalysis; Catechol 1,2-Dioxygenase; Dioxygenases/chemistry; Dioxygenases/genetics; Dioxygenases/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Histidine/genetics; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Lactones/chemistry; Lactones/metabolism; Mutagenesis, Site-Directed; Oxygenases/chemistry; Oxygenases/metabolism; Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/isolation & purification; Recombinant Fusion Proteins/metabolism |
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