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PDBsum entry 2eie
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Oxidoreductase
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PDB id
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2eie
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References listed in PDB file
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Key reference
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Title
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The stacking tryptophan of galactose oxidase: a second-Coordination sphere residue that has profound effects on tyrosyl radical behavior and enzyme catalysis.
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Authors
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M.S.Rogers,
E.M.Tyler,
N.Akyumani,
C.R.Kurtis,
R.K.Spooner,
S.E.Deacon,
S.Tamber,
S.J.Firbank,
K.Mahmoud,
P.F.Knowles,
S.E.Phillips,
M.J.Mcpherson,
D.M.Dooley.
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Ref.
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Biochemistry, 2007,
46,
4606-4618.
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PubMed id
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Abstract
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The function of the stacking tryptophan, W290, a second-coordination sphere
residue in galactose oxidase, has been investigated via steady-state kinetics
measurements, absorption, CD and EPR spectroscopy, and X-ray crystallography of
the W290F, W290G, and W290H variants. Enzymatic turnover is significantly slower
in the W290 variants. The Km for D-galactose for W290H is similar to that of the
wild type, whereas the Km is greatly elevated in W290G and W290F, suggesting a
role for W290 in substrate binding and/or positioning via the NH group of the
indole ring. Hydrogen bonding between W290 and azide in the wild type-azide
crystal structure are consistent with this function. W290 modulates the
properties and reactivity of the redox-active tyrosine radical; the Y272 tyrosyl
radicals in both the W290G and W290H variants have elevated redox potentials and
are highly unstable compared to the radical in W290F, which has properties
similar to those of the wild-type tyrosyl radical. W290 restricts the
accessibility of the Y272 radical site to solvent. Crystal structures show that
Y272 is significantly more solvent exposed in the W290G variant but that W290F
limits solvent access comparable to the wild-type indole side chain.
Spectroscopic studies indicate that the Cu(II) ground states in the semireduced
W290 variants are very similar to that of the wild-type protein. In addition,
the electronic structures of W290X-azide complexes are also closely similar to
the wild-type electronic structure. Azide binding and azide-mediated proton
uptake by Y495 are perturbed in the variants, indicating that tryptophan also
modulates the function of the catalytic base (Y495) in the wild-type enzyme.
Thus, W290 plays multiple critical roles in enzyme catalysis, affecting
substrate binding, the tyrosyl radical redox potential and stability, and the
axial tyrosine function.
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