Autoredox interconversion of two galactose oxidase forms GOase(ox) and GOase(semi) with and without dioxygen.
Solutions of galactose oxidase stored in air give in 3-4 h a mix of GOase(ox)(Cu(II)-Tyr(*)) and GOase(semi)(Cu(II)-Tyr), as a result of processes involving the formation and decay of the Cu(II)-coordinated tyrosyl radical (Tyr(*)). In this work the two reactions have been studied by UV-vis spectrophotometry and separate rate laws defined. The first involves the "spontaneous" autoreduction of GOase(ox) to GOase(semi), which in air-free conditions is 100% complete. Rate constants (k(red)) are dependent on pH, and previously defined acid dissociation constants pK(1a) = 5.7 (exogenous H(2)O ligand), and pK(2a) = 8.0 (axial H(+)Tyr-495) apply. Values of k(red)(25 degrees C) range from 1.55 x 10(-4) s(-1) (pH 5.5) to 2.69 x 10(-4) s(-1) (pH 8.6), I = 0.100 M (NaCl). No reaction occurs with N(3)(-) or NCS(-) present in amounts sufficient to give >98% binding at the substrate binding (exogenous) site, while CH(3)CO(2)(-) and phosphate (less extensively bound) also inhibit the reaction. From such inhibition studies K(25 degrees C) is 161 M(-1) at pH 6.4 for acetate (previous value 140 M(-)(1)) and 46 M(-1) at pH 7.0 for phosphate. No reaction occurs when the disulfide Cys515-Cys518 (10.2 A from the Cu) is chemically modified with HSPO(3)(2-), and electron transfer via the disulfide and exogenous position is proposed (source of the electron not established). The conversion of GOase(semi) to GOase(ox) only occurs with O(2) present, when a first-order dependence on [O(2)] is observed, giving k(ox)(25 degrees C) = 0.021 M(-1) s(-1) at pH 7.5. This process is unaffected by NCS(-) or N(3)(-) bound at the exogenous site, and a mechanism involving outer-sphere reaction of O(2) to O(2)(-) followed by a fast step O(2) to H(2)O(2) is proposed. As GOase(ox) is formed, autoreduction back to GOase(semi) occurs, and at pH 7.5 with O(2) in large excess (1.13 mM) the maximum conversion to GOase(ox) is 69%. The k(ox) reaction proceeds to completion with >98% N(3)(-) bound at the exogenous site.
Wright
C
C
Department of Chemistry, The University of Newcastle, Newcastle upon Tyne, NE1 7RU, UK.
Sykes
A G
AG
D005670:Fusarium / Q000201:enzymology; D005692:Galactose Oxidase / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D011487:Protein Conformation
D007527:Isoenzymes; D005692:Galactose Oxidase; D010100:Oxygen