Reactivity of galactose oxidase
| Autor: | Achim Sokolowski, Colin G. Saysell, A. Geoffrey Sykes, Craig Wright, Christopher D. Borman, MB Twitchett |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
biology
Substrate (chemistry) Active site Medicinal chemistry Acid dissociation constant Inorganic Chemistry chemistry.chemical_compound Reaction rate constant chemistry Galactose oxidase Radiolysis Materials Chemistry biology.protein Organic chemistry Enzyme kinetics Physical and Theoretical Chemistry Raffinose |
| Zdroj: | Coordination Chemistry Reviews. :771-779 |
| ISSN: | 0010-8545 |
| DOI: | 10.1016/s0010-8545(99)00120-4 |
| Popis: | Recent studies on reactions of the two-equivalent CuII/tyrosyl radical containing enzyme galactose oxidase (GOase) from Fusarium NRRL 2903 are referred to in this report. Two GOaseox active-site acid dissociation pKa values have been determined by UV–vis spectrophotometry, and are 5.7 (coordinated H2O) and 7.0 (protonated Tyr-495). The active enzyme (GOaseox) 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, GOaseox+RCH2OH→GOaseredH2+RCHO (k1) and GOaseredH2+O2→GOaseox+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 GOaseredH2. Michaelis–Menten kinetics allow Km (=1/Kbind) and kcat (catalytic turnover) to be determined, where Kbind 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 GOasesemi with CO2 − (which is unable to provide protons) are also described. These indicate that, after initial reduction to the CuI state (GOasered), 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 GOaseox also involves the disulfide. As a means of modelling substrate binding, NCS− substitution at the CuII active site of GOaseox 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 kf=1.1×104 M−1 s−1, I=0.100 M (NaCl). |
| Databáze: | OpenAIRE |
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