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Lookup NU author(s): Craig Wright, Dr Sang-choul Im, Mark Twitchett, Emeritus Prof Alfred Sykes
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Thermodynamic and kinetic studies on the X- = NCS-, N3-, and CH3CO2- replacement of H2O/OH- at the CuII exogenous site of the tyrosyl-radical-containing enzyme galactose oxidase (GOaseox) from Fusarium (NRR 2903), have been studied by methods involving UV - vis spectrophotometry (25 °C); pH range 5.5-8.7, I = 0.100 M (NaCl). In the case of N3- and CH3CO2- previous X-ray structures have confirmed coordination at the exogenous H2O/OH- site. From the effect of pH on the UV - vis spectrum of GOaseox under buffer-free conditions, acid dissociation constants of 5.7 (pK1a; coordinated H2O) and 7.0 (pK2a; H+Tyr-495) have been determined. At pH 7.0 formation constants K(25°C)/M-1 are NCS- (480), N3- (1.98 × 104), and CH3CO2- (104), and from the variations in K with pH the same two pKa values are seen to apply. No pK1a is observed when X- is coordinated. From equilibration stopped-flow studies rate constants at pH 7.0 for the formation reaction kf(25°C)/M-1 s-1 are NCS- (1.13 × 104) and N3- (5.2 × 105). Both K and kf decrease with increasing pH, consistent with the electrostatic effect of replacing H2O by OH-. In the case of the GOaseox Tyr495Phe variant pK1a is again 5.7, but no pK2a is observed, confirming the latter as acid dissociation of protonated Tyr-495. At pH 7.0, K for the reaction of four-coordinate GOaseox Tyr495Phe with NCS- (1.02 × 105M-1) is more favorable than the value for GOaseox. Effects of H+Tyr-495 deprotonation on K are smaller than those for the H2O/OH- change. The pK1a for GOasesemi is very similar (5.6) to that for GOaseox (both at CuII), but pK2a is 8.0. At pH 7.0 values of K for GOasesemi are NCS- (270 M-1), N3- (4.9 × 103), and CH3CO2- (107).
Author(s): Wright C, Im S-C, Twitchett MB, Saysell CG, Sokolowski A, Sykes AG
Publication type: Article
Publication status: Published
Journal: Inorganic Chemistry
Year: 2001
Volume: 40
Issue: 2
Pages: 294-300
ISSN (print): 0020-1669
ISSN (electronic): 1520-510X
Publisher: American Chemical Society
URL: http://dx.doi.org/10.1021/ic000162w
DOI: 10.1021/ic000162w
PubMed id: 11170534
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