title: Thermodynamic, kinetic and pH studies on the reactions of NCS-, N3-, and CH3CO2- with Fusarium galactose oxidase.
abstract: 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 degrees 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 degrees C)/M-1 are NCS- (480), N3- (1.98 x 10(4)), 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 degrees C)/M-1 s-1 are NCS- (1.13 x 10(4)) and N3- (5.2 x 10(5)). 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 x 10(5) M-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 x 10(3)), and CH3CO2- (107).
authors.affiliation: Department of Chemistry, University of Newcastle, Newcastle upon Tyne, NE1 7RU, UK.
authors.forename: C
authors.initials: C
authors.lastname: Wright
authors.forename: S C
authors.initials: SC
authors.lastname: Im
authors.forename: M B
authors.initials: MB
authors.lastname: Twitchett
authors.forename: C G
authors.initials: CG
authors.lastname: Saysell
authors.forename: A
authors.initials: A
authors.lastname: Sokolowski
authors.forename: A G
authors.initials: AG
authors.lastname: Sykes
mesh_terms: D002254:Carbonates / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005670:Fusarium / Q000201:enzymology*; D005692:Galactose Oxidase / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009584:Nitrogen / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013440:Sulfides / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D002254:Carbonates; D013440:Sulfides; D005692:Galactose Oxidase; D009584:Nitrogen