Mechanistic studies on the single copper tyrosyl- radical containing enzyme galactose oxidase

Studies on the single Cu protein galactose oxidase (68kDa; 639 amino acids) from Fusarium NRRL 2903 are described. The Cu is coordinated in a square based pyramid by Tyr-272, Tyr-495 (axial), His-496, His-581 and H20 (the substrate binding site), and the enzyme functions as a 2-equivalent oxidase, 0, + H,O,, with oxidation of primary alcohol substrates RCH20H to RCHO. The active enzyme has a coordinated tyrosyl (Tyr) free radical at Tyr-272, and along with the Cu" to Cu' redox change gives the required twoequivalent redox capacity. The three oxidation states are here written as GOase,, (Cu", Tyr,), GOase,, (Cu", Tyr), and GOase,,, (Cu', Tyr). Protonation of Tyr-495 is an important part of the enzymic reaction. Studies described are consistent with a mechanism involving H-atom transfer from substrate RCH20H to Tyr. at Tyr-272. INTRODUCTION AND GENERAL PROPERTIES Occurrence and Function Galactose oxidase (GOase; EC 1 .I .3.9) is a Type 2 single Cu enzyme secreted by a number of fungal species of which Fusarium NRRL 2903 formerly known as Polyporus circinatus and Dactylium dendroides has been the most extensively studied, (ref. 1-31, It serves as catalyst for the oxidation of a wide range of primary alcohols (RCH20H) including sugars, polysaccharides (in particular hexose derivatives), (ref. 2,4,5), in a twoequivalent oxidase action converting dioxygen to hydrogen peroxide. RCH,OH + 0, + RCHO + H202 (1 1 The reaction is stereospecific, (ref. 61, and the enzyme for example reacts with Dgalactose but not the L form. The oxidation states GOase,, (Cu", Tyr.1 and GOase,,, (Cu', Tyr) are involved in this change, (2). The intermediate state GOase,, (Cull, Tyr) although