Kinetic Studies on the Redox Interconversion of GOasesemi and GOaseox Forms of Galactose Oxidase with Inorganic Complexes as Redox Partners

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• 作者：Colin G. Saysell ; Christopher D. Borman ; Andrew J. Baron ; Michael J. McPherson ; and A. Geoffrey Sykes
• 刊名：Inorganic Chemistry
• 出版年：1997
• 出版时间：September 24, 1997
• 年：1997
• 卷：36
• 期：20
• 页码：4520 - 4525
• 全文大小：175K
• 年卷期：v.36,no.20(September 24, 1997)
• ISSN：1520-510X

文摘

Redox interconversions between the GOase_semi(Cu^II, Tyr) and tyrosyl radical containingGOase_ox (Cu^II, Tyr)oxidation states of the Cu-containing enzyme galactose oxidase (GOase)from Fusarium NRRL 2903 have beenstudied. The inorganic complexes[Fe(CN)₆]^3- (410 mV),[Co(phen)₃]³⁺ (370 mV),[W(CN)₈]^3- (530 mV),and[Co(dipic)₂]^- (362 mV)(E' values vs NHE; dipic = 2,6-dicarboxylatopyridine)were used as oxidants for GOase_semi,and [Fe(CN)₆]^4- and[Co(phen)₃]²⁺ as reductants forGOase_ox. On oxidation of GOase_semi aradical is generatedat the coordinated phenolate of Tyr-272 to give GOase_ox.The one-electron reduction potential E' (25 C)forthe GOase_ox/GOase_semi couple varies with pH andis 400 mV vs NHE at pH 7.5, the smallest value so farobservedfor a tyrosyl radical. The reactions are very sensitive to pH, ormore precisely to pK_a values ofGOase_semi andGOase_ox, and the charge on the inorganic reagent. Forexample, with [Fe(CN)₆]^3-as oxidant, the rate constant(25 C)/M^-1 s^-1of 0.16 × 10³ (pH ~ 9.5) increases to 4.3 ×10³ (pH ~ 5.5), while for[Co(phen)₃]³⁺ a valueof 4.9 × 10³ (pH ~ 9.5) decreases to 0.04 ×10³ (pH ~ 5.5), I = 0.100 M (NaCl).From the kinetics a singleGOase_semi acid dissociation process,pK_a = 8.0 (average), has been confirmed byUV-vis spectrophotometricstudies (7.9). The corresponding value for GOase_ox is6.7. No comparable kinetic or spectrophotometric pHdependences are observed with the Tyr495Phe variant, indicating theaxial Tyr-495 as the site of protonation.Neutral CH₃CO₂H and HN₃species bind at the substrate binding site of GOase_semi,thus mimicking the behaviorof primary alcohols RCH₂OH, the natural substrate ofGOase. On coordination, loss of a proton occurs, andinhibition of the oxidation with[Fe(CN)₆]^3- isobserved.