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Galactose Oxidase

Galactose oxidase is a monomeric enzyme that catalyzes the oxidation of primary alcohols to aldehydes and contains a unique thioether bond between tyrosine and cysteine. The enzyme's catalytic activity is influenced by specific residues, with mutations significantly reducing activity. Tryptophan-290 plays a crucial role in stabilizing the enzyme's active site and protecting the cofactor from solvent exposure.

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90 views7 pages

Galactose Oxidase

Galactose oxidase is a monomeric enzyme that catalyzes the oxidation of primary alcohols to aldehydes and contains a unique thioether bond between tyrosine and cysteine. The enzyme's catalytic activity is influenced by specific residues, with mutations significantly reducing activity. Tryptophan-290 plays a crucial role in stabilizing the enzyme's active site and protecting the cofactor from solvent exposure.

Uploaded by

alfredo.angeles
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© © All Rights Reserved
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Galactose Oxidase

Galactose Oxidase

• Galactose oxidase is a monomeric enzyme that contains a single copper ion and catalyses the
stereospecific oxidation of primary alcohols to their corresponding aldehydes
• The protein contains an unusual covalent thioether bond between a tyrosine, which acts as a radical center
during the two-electron reaction, and a cysteine
• The enzyme is produced in a precursor form lacking the thioether bond and also possessing an additional
17-aa pro-sequence at the N terminus

PNAS 2001, 98 (23) 12932-12937


Potential mechanisms for thioether bond formation
Galactose oxidase activity

Mutant Relative catalytic activity


WT 1.0
C228G 0.0001
W290H 0.0005
Y495F 0.001
Role of residues
• Tyrosine-495, the axial tyrosinate ligand in the copper complex, may function as a general base during catalysis.
This role has been proposed on the basis of spectroscopic evidence for displacement of the axial tyrosine in
anion complexes associated with uptake of a single proton
• The temperature dependence of the structure of the Cu(II) complex of WT enzyme, reflected in the ligand field (d
→ d) spectra of the metal ion has been interpreted as a conversion of an aquo complex with Tyr495 bound to a
hydroxide complex with Tyr495 displaced, mimicking the changes that occur on anion binding and defining a
putative proton-transfer coordinate in the active site involving Tyr495 and a cis-coordinated hydroxylic species

Mutant Relative catalytic activity


WT 1.0
C228G 0.0001
W290H 0.0005
Y495F 0.001
Role of residues

Mutant Relative catalytic activity


WT 1.0
C228G 0.0001
W290H 0.0005
Y495F 0.001

• Trp290 has attracted special attention because of its close proximity to the Tyr−Cys cross-link. The tryptophan
side chain lies parallel to the plane of the Tyr−Cys cofactor
• The tryptophan side chain is oriented such that the indole N−H group is approximately aligned with the Tyr272
phenolic C−O vector
• The stability of the active enzyme is reported to be significantly lower in the W290H mutant, supporting a role for
the active site tryptophan in shielding the Tyr−Cys cofactor from exposure to solvent
Galactose selectivity

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