Galactose Oxidases

Galactose Oxidase (GO) from the filamentous wheat-root fungus Fusarium spp. is a mononuclear type 2 copper enzyme that catalyzes the two-electron oxidation of a large number of primary alcohols to their corresponding aldehydes, coupled with the reduction of dioxygen to hydrogen peroxide [1,18]  

The protein is a single polypeptide with molecular mass of ca 68 kDa. To perform the two-electron chemistry, the enzyme utilizes, in addition to the copper center, a protein radical cofactor, which has been assigned to the Tyr272 residue. GO can exist in three distinct oxidation states the highest state with Cu(II) and tyrosyl radical, the intermediate state with Cu(II) and tyrosine, and the lowest state with Cu(I) and tyrosine. The highest oxidation state is the catalytically active one. The protein radical couples antiferromagnetically with the copper ion, resulting in an EPR silent species. 

F re 1. Crystal structure of the active site of galactose oxidase. The substrate 

In accordance with the general guidelines given above for the choice of chemical models, the two histidines were modeled by imidazoles, the equatorial tyrosine was modeled by SH-substituted phenol, whereas the somewhat smaller, but fully adequate, vinyl alcohol served as model for the axial tyrosine. The 

As in the case of PFL (see below), a charge-neutral model was used for galactose oxidase. This model implies that one of the histidine ligands needs to be deprotonated in order to obtain the correct oxidation state of the copper atom. 

1 Galactose oxidase. Galactose oxidase is a fungal enzyme that oxidises C6 of D-galactose to the corresponding aldehyde, reducing molecular 

In this review, current understanding of the structure and function of galactose oxidase and amine oxidases will be described together with comparisons between them and future directions in this field. 

FIGURE 1. Oxidation of D-galactose to aldehyde and hydrogen peroxide catalysed by GOase. 

The major purpose of this review is to describe studies on the structure and function of galactose oxidase carried out over the last decade. As with all science, these advances are based on many earlier studies and it will not be possible to document with due respect how these studies advanced the field. Earlier work (up to 1984) on GOase has been reviewed (Malm-strom et al., 1975 1981 Kosman, 1984, 13nl5) and more recent work is covered in reviews by Whittaker (1994) and by Knowles and Ito (1993). 

Specificity for the alcohol substrate is very broad, ranging from small molecules (e.g. propanediol) to polysaccharides. Galactose oxidase is strictly stereo specific. It does not oxidise either o-glucose or l-galactose. 

A covalent linkage between Tyr272 and Cys228 has been observed, whose functional role may relate to the presence of a tyrosine free radical at Tyr272. The tyrosine free radical could be stabilised by delocalization to Cys228 and stacking interactions with Tryp290 (Ito et al. 1994). 

The formally two-electron redox reactions, the alcohol oxidation and the O, reduction, are per- 

Hydrogen atom abstraction by Cu(II)- and Zn(II)-phenoxyl radical complexes was used as a model for the active form of galactose oxidase (Taki et al. 2000). 

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Copper is one of the twenty-seven elements known to be essential to humans (69—72) (see Mineral nutrients). The daily recommended requirement for humans is 2.5—5.0 mg (73). Copper is probably second only to iron as an oxidation catalyst and oxygen carrier in humans (74). It is present in many proteins, such as hemocyanin [9013-32-3] galactose oxidase [9028-79-9] ceruloplasmin [9031 -37-2] dopamine -hydroxylase, monoamine oxidase [9001-66-5] superoxide dismutase [9054-89-17, and phenolase (75,76). Copper aids in photosynthesis and other oxidative processes in plants. 

A number of complexes of copper with 1,1-dithiolenes are known they are interesting, inasmuch as they form (1) polynuclear species, e.g., [Cu4(i-mnt)3]2 . Recently, a copper(III) complex of 1,1-dicarboeth-oxy-2-ethylenedithiolate (DED ) was prepared (375) by oxidation of aqueous solutions of K2[Cu(DED)2] with a 10-15% excess of Cu(II) or H202, and of (BzPh3P)2[Cu(DED)2] with I2. The possibility of this system as a model for the Cu "/Cu. system in n-galactose oxidase has been pointed out. Lewis and Miller (113) also prepared M[Cu(S2C CHN02)2] (M = Cu, or Zn) and Cu[Cu S2C C(CN)2 2], and found that they are effective insecticides. 

Rogers MS, Dooley DM. 2001. Posttranslationally modihed tyrosines from galactose oxidase and cytochrome c oxidase. Adv Protein Chem 58 387. 

Copper(II) complexes with phenoxo ligands have attracted great interest, in order to develop basic coordination chemistry for their possible use as models for tyrosinase activity (dimeric complexes) and fungal enzyme galactose oxidase (GO) (monomeric complexes). The latter enzyme catalyzes the two-electron oxidation of primary alcohols with dioxygen to yield aldehyde and... 

Siebum, A., van Wijk, A., Schoevaart, R. and Kieboom, T. (2006) Galactose oxidase and alcohol oxidase scope and limitations for the enzymatic synthesis of aldehydes. Journal of Molecular Catalysis B-Enzymatic, 41 (3-1), 141-145. 

Fialova, R, Namdjou, D.-J., Ettrich, R. et al. (2005) Combined application of galactose oxidase and beta-iV-acetylhexosaminidase in the synthesis of complex immuno-active, V-acclyl-D-galaclosamiHides. Advanced Synthesis and Catalysis, 347 (7 + 8), 997-1006. 

Figure 1.103 Galactose oxidase may be used to transform specifically the C-6 hydroxyl group of galactose into an aldehyde.

Add 0.05 units of Vibrio cbolerae neuraminidase and 5 units of galactose oxidase per ml of cell suspension. 

To each ml of glycoprotein solution, add 30 pi of neuraminidase (1 unit/ml as supplied by Behringwerke AF), then 30 pi of galactose oxidase (previously dissolved at 100 units/ml in the labeling buffer of step 1), and finally 100 pi of the biocytin hydrazide solution. 

Fortier [6] found that AQ polymer from Eastman was not deleterious for the activity of a variety of enzymes such as L-amino acid oxidase, choline oxidase, galactose oxidase, and GOD. Following mixing of the enzyme with the AQ polymer, the mixture was cast and dried onto the surface of a platinum electrode. The film was then coated with a thin layer of Nafion to avoid dissolution of the AQ polymer film in the aqueous solution when the electrode was used as a biosensor. These easy-to-make amperometric biosensors, which were based on the amperometric detection of H202, showed high catalytic activity. 

By application of EMMA Regehr and Regnier developed several assays for enzymes that produce (galactose oxidase and glucose oxidase) or consume (catalase) hydrogen peroxide. Unlabeled enzymes were determined in the femto-mole mass range, while detection limits of less than 10,000 molecules were reported for catalase [101]. 

The immunological properties of a modified S14, obtained on treatment with D-galactose oxidase and subsequent oxidation of the aldehyde group to a carboxyl group with chlorite, have been investigated.45... 

E S R.. Fenton. Transferrin Complexes with Non-Physiological and Toxic Metals, David M. Taylor. Transferrins, Edward N. Baker. Galactose Oxidase, Peter Knowles and Nobutoshi Ito. Chemistry of Aqua Ions of Biological Importance, David T. Richens. From a Structural Perspective Structure and Function of Manganese - Containing Biomolecules, David C. Weatherburn, Index. Volume 3,1996,304 pp. 109.50/ 70.00 ISBN 1-55938-642-8... 

Nonblue oxidases Amine oxidase Diamine oxidases Galactose oxidase Cytochrome c oxidase... 

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