Circular dichroism spectra of the copper enzyme, galactose oxidase, in the presence of its substrates and products

Ettinger, Murray J.; Kosman, Daniel J.

doi:10.1021/bi00703a030

Cited by 12 publications

(16 citation statements)

References 10 publications

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“…The Tyr495Phe variant was obtained in a similar manner. Both WT and Tyr495Phe GOase concentrations were determined from the absorbance at 280 nm ( p1.05!10 5 M P1 cm P1 ) [19], which is independent of oxidation state and pH. GOase ox is green in colour, and GOase semi a less intense blue.…”

Section: Enzyme Solutionsmentioning

confidence: 99%

Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

1997

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Reactions (25 7C) of galactose oxidase, GOase ox from Fusarium NRRL 2903 with five different primary-alcohol-containing substrates RCH 2 OH:-D-galactose (I) and 2-deoxy-D-galactose (II) (monosaccharides); methyl-b-D-galactopyranoside (III) (glycoside); D-raffinose (IV) (trisaccharide); and dihydroxyacetone (V) have been studied in the presence of O 2 . The GOase ox state has a tyrosyl radical coordinated at a square-pyramidal Cu II active site, and is a two-equivalent oxidant. Reactant concentrations were [GOase ox ] (0.8-10 mM), RCH 2 OH (1.0-6.0 mM), and O 2 (0.14-0.29 mM), with Ip0.100 M (NaCl). The reactions, monitored at 450 nm by stopped-flow spectrophotometry, terminated with depletion of the O 2 . Each trace was fitted to the competing reactions GOase ox cRCH 2 OH ] GOase red H 2 cRCHO (k 1 ), and GOase red H 2 cO 2 ] GOase ox cH 2 O 2 (k 2 ), with GOase red H 2 written as the doubly protonated two-electron-reduced Cu I product. It was necessary to avoid auto-redox interconversion of GOase ox and GOase semi . Information obtained at pH 7.5 indicates a 5 : 95 (ox : semi) "native" mix equilibration complete in F3 h. At pH `7.5, rate constants 10 P4 k 1 /M P1 s P1 for the reactions of GOase ox with (I) (1.19), (II) (1.07), (III) (1.29), (IV) (1.81), (V) (2.94) were determined. On decreasing the pH to 5.5, k 1 values decreased by factors of up to a half, and acid dissociation pK a s in the range 6.6-6.9 were obtained. UVVis spectrophotometric studies on GOase ox gave an independently determined pK a of 6.7. No corresponding reactions of the Tyr495Phe variant were observed, and there are no similar UV-Vis absorbance changes for this variant. The pK a is therefore assigned to protonation of Tyr-495 which is a ligand to the Cu. The rate constant k 2 (1.01!10 7 M P1 s P1 ) is independent of pH in the range 5.5-9.0 investigated, suggesting that H c (or H-atoms) for the O 2 ] H 2 O 2 change are provided by the active site of GOase red . The Cu I of GOase red is less extensively complexed, and a coordination number of three is likely.

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Section: Enzyme Solutionsmentioning

confidence: 99%

Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

1997

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“…Slit widths were programmed to maintain a constant 15-Á bandwidth, solutions for binding studies were prepared as previously described (Ettinger and Kosman, 1974). Spectra given in figures are averages from two to five separate experiments.…”

Section: Methodsmentioning

confidence: 99%

“…Fluorescence is also markedly affected by removal of the copper atom (Weiner et al, 1977). Furthermore, dihydroxyacetone, which is an excellent substrate, and the galacto-hexodialdose product both have profound effects on the tryptophan optical activity exhibited by galactose oxidase (Ettinger and Kosman, 1974). While it is difficult to imagine how an indole moiety could directly act as a copper ligand, the results reported herein demonstrate that oxidation of probably a single tryptophan residue in galactose oxidase has profound effects on both the catalytic activity and spectral properties of the Cu(II) atom in galactose oxidase.…”

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confidence: 99%

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

Kosman¹,

Ettinger²,

Bereman³

et al. 1977

Biochemistry

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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.

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“…orbital, making electron paramagnetic resonance (EPR) possible, as first shown by Gordy and Rexrod. 6 The heme structures in HbNO and a free model oxyheme are very similar:7"8 the FeNO and FeOO bond angles are less than 180°and in both structures more than one type of diatomic molecule projection on the heme (6) Gordy, W.; Rexrod, . N. In "Free Radicals in Biological Systems", Blois, M. S., Ed., Academic Press: New York; 1961.…”

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confidence: 98%

“…Marwedel, submitted 0 Reference 14. 6 Reference 11. c Reference 13. d A^i s the isotropic coupling constant; see ref 13. eAa = 14 ||-Ay\is the anisotropic coupling constant; see ref 13. ?…”

mentioning

confidence: 99%

Magnetic resonance studies of cyanide and fluoride binding to galactose oxidase copper(II): evidence for two exogenous ligand sites

Marwedel¹,

Kosman²,

Bereman³

et al. 1981

J. Am. Chem. Soc.

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Circular dichroism spectra of the copper enzyme, galactose oxidase, in the presence of its substrates and products

Cited by 12 publications

References 10 publications

Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

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

Magnetic resonance studies of cyanide and fluoride binding to galactose oxidase copper(II): evidence for two exogenous ligand sites

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