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PDBsum entry 1lj1
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Oxidoreductase
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PDB id
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1lj1
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Engineering water to act as an active site acid catalyst in a soluble fumarate reductase.
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Authors
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C.G.Mowat,
K.L.Pankhurst,
C.S.Miles,
D.Leys,
M.D.Walkinshaw,
G.A.Reid,
S.K.Chapman.
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Ref.
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Biochemistry, 2002,
41,
11990-11996.
[DOI no: ]
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PubMed id
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Abstract
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The ability of an arginine residue to function as the active site acid catalyst
in the fumarate reductase family of enzymes is now well-established. Recently, a
dual role for the arginine during fumarate reduction has been proposed [Mowat,
C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P.,
Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40,
12292-12298] in which it acts both as a Lewis acid in transition-state
stabilization and as a Brønsted acid in proton delivery. This proposal has led
to the prediction that, if appropriately positioned, a water molecule would be
capable of functioning as the active site Brønsted acid. In this paper, we
describe the construction and kinetic and crystallographic analysis of the Q363F
single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the
soluble fumarate reductase from Shewanella frigidimarina. Although replacement
of the active site acid, Arg402, with alanine has been shown to eliminate
fumarate reductase activity, this phenomenon is partially reversed by the
additional substitution of Gln363 with phenylalanine. This Gln --> Phe
substitution in the inactive R402A mutant enzyme was designed to "push" a water
molecule close enough to the substrate C3 atom to allow it to act as a Brønsted
acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme
does indeed reveal the introduction of a water molecule at the correct position
in the active site to allow it to act as the catalytic proton donor. The 1.8 A
resolution crystal structure of the Q363F mutant enzyme shows a water molecule
similarly positioned, which can account for its measured fumarate reductase
activity. However, in this mutant enzyme Michaelis complex formation is impaired
due to significant and unpredicted structural changes at the active site.
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