Role of tryptophan in the spectral and catalytic properties of the copper enzyme, galactose oxidase.

Previous results indicate that a tryptophan residue(s) may interact with the sugar substrate and Cu(II) atom of galactose oxidase (Ettinger, M. J., and Kosman, D. J. (1974), Biochemistry 13, 1248). We now show that N-bromosuccinimide (NBS) reduces enzymatic activity to 2% as two tryptophans are oxidized; only four residues are easily oxidized in the holoenzyme. An enzymatic activity vs. number of residues oxidized profile suggests that this inactivation is probably associated with only one of the first 2 residues oxidized. There is no evidence for chain cleavage or modification of amino acids other than tryptophan. While substrate protection is not afforded by the sugar substrate, the activity-related tryptophan is placed within the active-site locus by spectral evidence. NBS oxidation of two tryptophans results in a marked diminution of the large copper optical-activity transition at 314 nm. Under some reaction conditions, a doubling of ellipticity in the 600-nm region of copper CD is also observed. The effects of the NBS oxidation on the CD spectra of galactose oxidase permit the assignment of the 314-nm CD band to a charge-transfer transition and the 229-nm extremum to a specific tryptophan contribution. The AZZ parameter from electron spin resonance spectra is also markedly reduced by the NBS oxidation. Moreover, while cyanide binds to the native enzyme without reducing the Cu(II) atom, cyanide rapidly reduces the Cu(II) atom to Cu(I) in the NBS-oxidized enzyme. These CD and ESR results are taken to suggest that one aspect of the inactivation by NBS oxidation may be a conversion of the pseudosquare planar copper complex in the native enzyme to a more distorted, towards tetrahedral, complex in the inactivated enzyme. Since the inactivation can be accomplished without affecting binding of the sugar substrate, tryptophan oxidation must affect catalysis per se.     

The effect of potassium ferricyanide and copper (II) ions on the properties of immobilized galactose oxidase

Preparations of galactosooxidase (EC 1.1.3.9) immobilized by activated aminorganosilica have been used to study potassium ferricyanide and bivalent copper ions on the enzyme activity and stability in continuous reactor under pulse conditions. Introduction of potassium ferricyanide is shown to activate the enzyme and inconsiderably affecting its stability with the substrate absent and inducing inactivation of galactosooxidase in the process of catalytic reaction. Cu2+ ions, exerting no effect on the activity of immobilized galactosooxidase, evoke the enzyme inactivation in the process of catalysis.     

An investigation of the role of the copper in galactose oxidase.

Galactose oxidase is a metalloenzyme containing a single copper atom per molecule. The mechanism of action of galactose oxidase is studied in this paper by investigating substrate specificity and activation by peroxidase, and probing the copper site by electron spin resonance (ESR) spectroscopy. Line-shape simulation of ESR spectra are also reported and a comparison is made between observed and simulated spectra for galactose oxidase. A comparison is also reported for the enzyme from various commercial sources and enzyme isolated from a fungus in this laboratory. The results of this investigation suggest that the copper is in an environment of four in-plane nitrogens with axial symmetry.     

The radical chemistry of galactose oxidase

Galactose oxidase is a free radical metalloenzyme containing a novel metalloradical complex, comprised of a protein radical coordinated to a copper ion in the active site. The unusually stable protein radical is formed from the redox-active side chain of a cross-linked tyrosine residue (Tyr-Cys). Biochemical studies on galactose oxidase have revealed a new class of oxidation mechanisms based on this free radical coupled-copper catalytic motif, defining an emerging family of enzymes, the radical-copper oxidases. Isotope kinetics and substrate reaction profiling have provided insight into the elementary steps of substrate oxidation in these enzymes, complementing structural studies on their active site. Galactose oxidase is remarkable in the extent to which free radicals are involved in all aspects of the enzyme function: serving as a key feature of the active site structure, defining the characteristic reactivity of the complex, and directing the biogenesis of the Tyr-Cys cofactor during protein maturation.     

Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase

Directed evolution has become an important enabling technology for the development of new enzymes in the chemical and pharmaceutical industries. Some of the most interesting substrates for these enzymes, such as polymers, have poor solubility or form highly viscous solutions and are therefore refractory to traditional high-throughput screens used in directed evolution. We combined digital imaging spectroscopy and a new solid-phase screening method to screen enzyme variants on problematic substrates highly efficiently and show here that the specific activity of the enzyme galactose oxidase can be improved using this technology. One of the variants we isolated, containing the mutation C383S, showed a 16-fold increase in activity, due in part to a 3-fold improvement in K(m). The present methodology should be applicable to the evolution of numerous other enzymes, including polysaccharide-modifying enzymes that could be used for the large-scale synthesis of modified polymers with novel chemical properties.     

Role of carbohydrate content on the properties of galactose oxidase from Dactylium dendroides

The stability of intracellular, extracellular, and deglycosylated forms of galactose oxidase was compared with respect to the denaturing effects of heat, pH, and guanidine hydrochloride. The highly glycosylated forms were found to be more stable to pH and thermal inactivation. All forms were reversibly denaturated by guanidine hydrochoride, but the extent was dependent on the carbohydrate content. Deglycosylation did not affect the affinity of the enzyme for dihydroxyacetone and galactose. Exposure of different forms of galactose oxidase to proteases like pronase and trypsin resulted in a rapid degradation of the glycoenzymes with the formation of stable products. After pronase digestion of intra- and extracellular forms of galactose oxidase catalytic species were isolated by gel filtration. The species (61 and 42 kDa) isolated from pronase-digested extracellular enzyme lost their ability to oxidize primary alcohols. Species (67 and 46 kDa) obtained from the intracellular enzyme kept the specificity of the original enzyme. Active pronase-derived peptides (42 and 46 kDa, respectively) had a higher carbohydrate content than the inactive ones.     

Cofactor processing in galactose oxidase

GO (galactose oxidase; E.C. 1.1.3.9) is a monomeric 68 kDa enzyme that contains a single copper ion and an amino acid-derived cofactor. The enzyme is produced by the filamentous fungus Fusarium graminearum as an extracellular enzyme. The enzyme has been extensively studied by structural, spectroscopic, kinetic and mutational approaches that have provided insight into the catalytic mechanism of this radical enzyme. One of the most intriguing features of the enzyme is the post-translational generation of an organic cofactor from active-site amino acid residues. Biogenesis of this cofactor involves the autocatalytic formation of a thioether bond between Cys-228 and Tyr-272, the latter being one of the copper ligands. Formation of this active-site feature is closely linked to the loss of an N-terminal 17 amino acid prosequence. When copper and oxygen are added to this pro-form of GO (pro GO), purified in copper-free conditions from the heterologous host Aspergillus nidulans, mature GO is formed by an autocatalytic process. Structural comparison of pro GO with mature GO reveals overall structural similarity, but with some regions showing significant local differences in main-chain position. Some side chains of the active-site residues differ significantly from their positions in the mature enzyme. These structural effects of the prosequence suggest that it may act as an intramolecular chaperone to provide an open active-site structure conducive to copper binding and chemistry associated with cofactor formation. The prosequence is not mandatory for processing, as a recombinant form of GO lacking this region and purified under copper-free conditions can also be processed in an autocatalytic copper- and oxygen-dependent manner.     

Properties of Fusarium graminearum galactose oxidase

The kinetics and action mechanism of the galactose oxidase from Fusarium graminearum were studied. pH-optimum of the enzyme activity and stability was 7.0, the activity and stability of the galactose oxidase being decreased at any other values of pH. The enzyme is destabilized at acidic pH that is connected with protonization of its ionogenic group with pK 4.7. The temperature optimum of the galactose oxidase is 35 degrees C. When studying the enzyme thermoinactivation, it was found that at temperatures below 30 degrees C the energy of activation of denaturation was about 40 kcal/mole and at temperatures ranging from 30 to 70 degrees C - 13 kcal/mole. On the basis of the data obtained it was concluded that a low-temperature form of the galactose oxidase, possessing a higher energy of activation of denaturation, is more active than a high-temperature form. The value of Km for the enzyme in respect to galactose was 0.19 M, and the value of Vmax = 360 mumole/min per g of the preparation.     

Kinetic and magnetic resonance studies of substrate binding to galactose oxidase copper(II)

Alcohol substrate binding to the copper-containing enzyme galactose oxidase (GOase) has been studied by kinetic competition against cyanide and fluoride, 13C nmr relaxation, and esr competition experiments. The 13C nmr spectra of the substrate beta-O-methyl-D-galactopyranoside (beta-O-me-gal) show no apparent paramagnetic relaxation rate enhancement that could be attributed to innersphere equatorial binding of this molecule at the Cu(II) center. Moreover, the kinetics observed when CN- or F- are used as inhibitors of GOase with beta-O-me-gal as the substrate suggest that these anions act as apparent non-competitive inhibitors; the binding of the substrates beta-O-me-gal and O2 is not hindered per se, but the catalytic activity of the enzyme substrate complex is greatly decreased. The esr competition data also confirm that, in the absence of O2, CN- and beta-O-me-gal do not compete for the same GOase binding site. Previously reported esr and 19F nmr data show that CN- binds to the GOase Cu(II) at an equatorial coordination site, as does the F- detected in esr experiments. Thus, the results from the various competition experiments supports a model in which alcohol substrates bind outersphere to the GOase Cu(II), or, possibly, to an axial site.     

Dynamic and structural properties of glucose oxidase enzyme

The catalytic oxidation of β-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I′ band, tryptophan fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH₂ does not change the amount of α-helix present in the protein outer shell, but reorganises a fraction of random coil to β-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography, these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism. Received: 27 December 1996 / Accepted: 18 July 1997     

Exposure to galactose oxidase of GM1 ganglioside molecular species embedded into phospholipid vesicles

The exposure of GM1 molecular species present in the native ganglioside, carrying C18:1 or C20:1 long-chain bases (LCB), to Dactylium dendroides galactose oxidase was studied. When native GM1 (49.3% C18:1 and 50.7% C20:1 LCB, respectively), was inserted in dipalmitoylphosphatidylcholine vesicles and partially oxidized (10%), the proportion of C18:1 and C20:1 species in the oxidized GM1 was 59.6% and 40.4%, respectively, suggesting a preferential action of the enzyme on the shorter species. The V_max of the enzyme was higher on C18:1 GM1 than on C20:1 GM1. The molecular species were affected without any preference after partial (10%) oxidation of GM1 incorporated in egg phosphatidylcholine vesicles or in micellar form. These data indicate that the exposure of the terminal galactose moiety of GM1 ganglioside to galactose oxidase is affected by the ganglioside ceramide composition as well as the phospholipid environment, that presumably determine the distribution (molecular dispersion, segregation) of the ganglioside within the membrane.     

Various properties of galactose oxidase from Fusarium graminearum IMV-F-1060 immobilized on aminoorganosilochromes

Galactose oxidase preparations are obtained from Fusarium graminearum IMV-F-N 1060 immobilized on aminoorganosilochromes activated by cyanuron chloride and 2.4-toluylene diizocyanate. The immobilized preparations were studied for their selective action on different carbohydrate substrates and for the pH-medium dependence of the obtained preparation activity. Potassium ferricyanide is established to have an activating effect on the immobilized enzyme. It is shown that the immobilized galactose oxidase preparations may be used for the analysis of galactose and lactose.     

A first model for the oxidized active form of the active site in galactose oxidase: a free-radical copper complex

 Copper(II) complexes derived from the tripodal ligand bis(3′-t–butyl-2′-hydroxybenzyl)(2-pyridylmethyl)amine (LH₂) have been studied in order to mimic the redox active site of the free radical-containing copper metalloenzyme galactose oxidase. In non-coordinating solvents such as dichloromethane, only an EPR-silent dimeric complex was obtained (L₂Cu₂). The crystal structure of L₂Cu₂ revealed a "butterfly" design of the [Cu(μOR)₂Cu] unit, which is not flattened and leads to a short Cu–Cu distance, the t–butyl groups being localized on the same side of the [Cu(μOR)₂Cu] unit. The dimeric structure was broken down by acetonitrile or by alcohols, leading quantitatively to a brown mononuclear copper(II) complex. UV-visible and EPR data indicated the coordination of the solvent in these mononuclear complexes. Electrochemical as well as chemical (silver acetate) one-electron oxidation of acetonitrile solutions of the monomeric complex led to a yellow-green solution. Based on EPR, UV-visible and resonance Raman spectroscopy, the one-electron oxidation product was identified as a cupric phenoxyl radical system. It slowly decomposes into a product where the ligand has been substituted (dimerization) in the para position of the hydroxyl group, for one of the phenolic groups. The data for the one-electron oxidized species provides strong evidence for a free-radical copper (II) complex. Received: 19 July 1996 / Accepted: 16 October 1996     

Oxidation of glycolipids in liposomes by galactose oxidase

Small unilamellar phosphatidylcholine vesicles containing globo-series glycolipids were labeled by the galactose oxidase/NaB[3H]4 procedure. The major glycolipid of human red cells, globoside, was the best substrate for galactose oxidase both in vesicles and in tetrahydrofuran-containing buffer. The oxidation rates of membrane-bound ceramide trihexoside and Forssman glycolipid were one-fourth and one-tenth, respectively, of the oxidation rate of globoside. Membrane-bound ceramide dihexoside was not a substrate for galactose oxidase, although it was readily oxidized in tetrahydrofuran-containing buffer. Soluble sialoglycoproteins and membrane-incorporated glycophorin A stimulated the oxidation of globoside-containing vesicles, whereas membrane-bound GD1a ganglioside had no effect on globoside oxidation.