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Reactivity of galactose oxidase

Lookup NU author(s): Mark Twitchett, Craig Wright, Emeritus Prof Alfred Sykes

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Abstract

Recent studies on reactions of the two-equivalent Cu(II)/tyrosyl radical containing enzyme galactose oxidase (GOase) from Fusarium NRRL 2903 are referred to in this report. Two GOase(ox) active-site acid dissociation pK(a) values have been determined by UV-vis spectrophotometry, and are 5.7 (coordinated H2O) and 7.0 (protonated Tyr-495). The active enzyme (GOase(ox)) catalyses the oxidation of the primary alcohols RCH2OH + O2→ RCHO+ H2O2, where previous studies with five different substrates are extended to include saturation kinetics for D-Galactose and D-Raffinose. Two competing steps, GOase(ox) + RCH2OH → GOase(red)H2 + RCHO (k1) and GOase(red)H2 + O2 → GOase(ox) + H2O2 (k2) are observed. Rate constants k1 are dependent on pH, whereas k2 is independent of pH in the range 5.5-9.0. The k2 behaviour suggests that the two protons required to bring about the O2 → H2O2 conversion are provided by the protonated form GOase(red)H2. Michaelis-Menten kinetics allow K(m) (= 1/K(bind)) and k(cat) (catalytic turnover) to be determined, where K(bind) M-1 values are small for D-Galactose (6.7) and D-Raffinose (14.3), in keeping with the enzyme being extracellular. Pulse radiolysis studies on GOase(semi) with CO2/(·-) (which is unable to provide protons) are also described. These indicate that, after initial reduction to the Cu1 state (GOase(red)), a spontaneous decay of the unstable product occurs with formation of a disulfide radical anion (RSSR(·-)) detected by its absorbance at 450 nm. Slow decay of the disulfide radical is observed in a further step. Evidence obtained suggests that the spontaneous decay of the tyrosyl radical of GOase(ox) also involves the disulfide. As a means of modelling substrate binding, NCS- substitution at the Cu(II) active site of GOase(ox) has been investigated. Dependence on pH is again observed and at 25°C, pH 7.0 (10 mM phosphate), K= 0.5 103 M-1, with forward rate constant k(f)= 1.1 104 M-1 s-1, I=0.100 M (NaCl). (C) 1999 Elsevier Science S.A.


Publication metadata

Author(s): Borman CD, Saysell CG, Sokolowski A, Twitchett MB, Wright C, Sykes AG

Publication type: Conference Proceedings (inc. Abstract)

Publication status: Published

Conference Name: 33rd International Conference on Coordination Chemistry

Year of Conference: 1999

Pages: 771-779

ISSN: 0010-8545

Publisher: Elsevier

URL: http://dx.doi.org/10.1016/S0010-8545(99)00120-4

DOI: 10.1016/S0010-8545(99)00120-4

Library holdings: Search Newcastle University Library for this item

Series Title: Coordination Chemistry Reviews (Part II)

ISBN: 18733840


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