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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)
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UNSPECIFIED (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 (42). pp. 13390-13396. doi:10.1021/bi048518 ISSN 0006-2960.
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Official URL: http://dx.doi.org/10.1021/bi048518
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 Ill extradiol dioxygenases, positioned within 4-5 Angstrom 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.
Item Type: | Journal Article | ||||
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Subjects: | Q Science > QD Chemistry | ||||
Journal or Publication Title: | BIOCHEMISTRY | ||||
Publisher: | AMER CHEMICAL SOC | ||||
ISSN: | 0006-2960 | ||||
Official Date: | 26 October 2004 | ||||
Dates: |
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Volume: | 43 | ||||
Number: | 42 | ||||
Number of Pages: | 7 | ||||
Page Range: | pp. 13390-13396 | ||||
DOI: | 10.1021/bi048518 | ||||
Publication Status: | Published |
Data sourced from Thomson Reuters' Web of Knowledge
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