Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

Abstract

Reactions (25 7C) of galactose oxidase, GOase ox from Fusarium NRRL 2903 with five different primary-alcohol-containing substrates RCH 2 OH:-D-galactose (I) and 2-deoxy-D-galactose (II) (monosaccharides); methyl-b-D-galactopyranoside (III) (glycoside); D-raffinose (IV) (trisaccharide); and dihydroxyacetone (V) have been studied in the presence of O 2 . The GOase ox state has a tyrosyl radical coordinated at a square-pyramidal Cu II active site, and is a two-equivalent oxidant. Reactant concentrations were [GOase ox ] (0.8-10 mM), RCH 2 OH (1.0-6.0 mM), and O 2 (0.14-0.29 mM), with Ip0.100 M (NaCl). The reactions, monitored at 450 nm by stopped-flow spectrophotometry, terminated with depletion of the O 2 . Each trace was fitted to the competing reactions GOase ox cRCH 2 OH ] GOase red H 2 cRCHO (k 1 ), and GOase red H 2 cO 2 ] GOase ox cH 2 O 2 (k 2 ), with GOase red H 2 written as the doubly protonated two-electron-reduced Cu I product. It was necessary to avoid auto-redox interconversion of GOase ox and GOase semi . Information obtained at pH 7.5 indicates a 5 : 95 (ox : semi) "native" mix equilibration complete in F3 h. At pH `7.5, rate constants 10 P4 k 1 /M P1 s P1 for the reactions of GOase ox with (I) (1.19), (II) (1.07), (III) (1.29), (IV) (1.81), (V) (2.94) were determined. On decreasing the pH to 5.5, k 1 values decreased by factors of up to a half, and acid dissociation pK a s in the range 6.6-6.9 were obtained. UVVis spectrophotometric studies on GOase ox gave an independently determined pK a of 6.7. No corresponding reactions of the Tyr495Phe variant were observed, and there are no similar UV-Vis absorbance changes for this variant. The pK a is therefore assigned to protonation of Tyr-495 which is a ligand to the Cu. The rate constant k 2 (1.01!10 7 M P1 s P1 ) is independent of pH in the range 5.5-9.0 investigated, suggesting that H c (or H-atoms) for the O 2 ] H 2 O 2 change are provided by the active site of GOase red . The Cu I of GOase red is less extensively complexed, and a coordination number of three is likely.

Galactose Oxidase as a Model for Reactivity at a Copper Superoxide Center

“…The values for k cat and K m (H 2 -OMeGal), Table 1, are in agreement with literature reports of 1180 s −1 and 175 mM for reaction of GAOX with galactose at 20 °C and pH 7.0 24. The value for k cat /K m (H 2 -OMeGal) compares favorably with reported second-order rate constants in the range of 1 × 10 4 M −1 s −1 3,4. The much faster value of k cat /K m (O 2 ), 2.6(0.3) × 10 6 m −1 s −1 , is also in the range of previously reported values for second-order rate constants of 8 × 10 6 M −1 s −1 at 4 °C and 1 × 10 7 M −1 s −1 at 25 °C 3,4.…”

“…The value for k cat /K m (H 2 -OMeGal) compares favorably with reported second-order rate constants in the range of 1 × 10 4 M −1 s −1 3,4. The much faster value of k cat /K m (O 2 ), 2.6(0.3) × 10 6 m −1 s −1 , is also in the range of previously reported values for second-order rate constants of 8 × 10 6 M −1 s −1 at 4 °C and 1 × 10 7 M −1 s −1 at 25 °C 3,4. However, prior efforts to obtain K m (O 2 ) for galactose oxidase were unsuccessful and it was concluded that the enzyme could not be saturated with oxygen 24,25.…”

“…As illustrated in Figure 1, reciprocal plots of k (app) vs. [O 2 ] at varying concentrations of H 2 -OMeGal show close to parallel lines, consistent with a steady-state ping-pong mechanism wherein the reductive and oxidative reactions half-reactions (Scheme 1) are separated by an irreversible step. A ping-pong mechanism was predicted for GAOX on the basis of stopped flow experiments,3,4 but had not been previously shown under steady-state conditions. The values for k cat and K m (H 2 -OMeGal), Table 1, are in agreement with literature reports of 1180 s −1 and 175 mM for reaction of GAOX with galactose at 20 °C and pH 7.0 24.…”

“…GAOX catalyzes the oxidation of a broad range of primary alcohols to their corresponding aldehyde. Stopped flow studies have demonstrated that the net reaction can be separated into two half-reactions,3,4 the first of which involves substrate oxidation and reduction of the enzyme to yield Cu(I)-tyrosinate, eq. (1): $$\mathrm{E}-\mathrm{B}{:}^{-}+{\mathrm{Cu}}^{\mathrm{II}}-(•\mathrm{normalO}-\mathrm{TyrCys})+{\mathrm{RCH}}_2\mathrm{OH}\to \mathrm{E}-\mathrm{BH}+{\mathrm{Cu}}^{\mathrm{normalI}}-(\mathrm{HO}-\mathrm{TyrCys})+\mathrm{RCHO}$$ This process can be further broken down into three steps: proton transfer from substrate to an active site base, hydrogen atom transfer to the cofactor-based radical center, and electron transfer to the Cu(I), Scheme 1A.…”

Probing structural and catalytic characteristics of galactose oxidase confined in nanoscale chemical environments

“…The sample solution was stirred at 480 rpm and the electrode calibrated in air-saturated Milli-Q water (z 0.252 mM O 2 ). 50 In each measurement, air bubbles were excluded before the start of the measurement. 50 ml of free or immobilized enzyme (stored on ice) was injected into a 3 ml reaction mixture of air-saturated 5-400 mM galactose and 1 mM K 3 [Fe(CN) 6 ] in buffer (pH 7.0) at 25 C through the access slot using a syringe.…”