β-Haloethanol Substrates as Probes for Radical Mechanisms for Galactose Oxidase

Abstract

Ketyl radical anions with a halogen substitutent on the carbon adjacent to ketyl are known to rapidly rearrange by halide anion ejection. Such a rearrangement is an ideal probe for possible ketyl radical anion intermediates in the catalytic mechanism of the monocopper/tyrosine radical enzyme galactose oxidase (GOase). Turnover of β-fluoro-, β-chloro-, β-bromo-, and β-iodoethanol by GOase leads to mechanism-based inactivation of the enzyme by trapping the enzyme in a catalytically inactive one-electron-reduced form. Presuming that mechanism-based inactivation and turnover proceed through the same reactive intermediates, the data reported here narrow down the possible mechanisms for the substrate oxidation step (the two electron transfer from substrate to enzyme) to two similar possibilities. Either the reaction proceeds through a short-lived ketyl radical anion intermediate or it proceeds through a closely related concerted E2R mechanism with considerable ketyl radical anion character in the transition state.

Theoretical studies on the reaction mechanism of oxidation of primary alcohols by Zn/Cu(ii)-phenoxyl radical catalyst

“…4g However, on the basis of the conclusion made by Wachter et al using b-haloethanol as the mechanism probe, "the substrate oxidation step of GOase proceeds either through a short-lived ketyl radical anion intermediate or through a closely related E 2 R mechanism with considerable ketyl radical anion character in the transition state". 19 Therefore, in the present work, two different substrate oxidation steps for Cu-catalyst are shown in Scheme 2, i.e., the concerted radical mechanism with transition state in a considerable ketyl radical anion character (Scheme 2A) and step-wise radical mechanism with a ketyl radical anion intermediate (Scheme 2B). Despite several attempts, no ketyl radical intermediate could be found in our calculations.…”

“…Despite several attempts, no ketyl radical intermediate could be found in our calculations. Therefore, the results of the DFT calculation combined with the conclusion from Wachter et al 19 suggest that the substrate oxidation step of the Cu-catalyst is the concerted radical mechanism with a radical character transition state 3 2-3. After the hydrogen atom transfer, 3 is generated.…”

Radical copper oxidases, one electron at a time

“…The essential proton transfer and redox functions built into this active site define the key features of a catalytic mechanism for substrate oxidation. Although the structure of substrate complexes of galactose oxidase are unknown aside from model-building predictions [5,30], conservative replacement of the coordinated solvent hydroxyl with alcohol hydroxyl seems fully justified by the evidence for exogenous ligand interactions at the solvent site, with the additional stereochemical constraint of stereospecific pro-S methylene hydrogen abstraction in the oxidation step. This leads to a picture of proton transfer from the acidified hydroxyl of the coordinated alcohol to tyrosine Y495 phenolate tri gering substrate activation [1,17].…”

Homemade cofactors: Self-processing in galactose oxidase

“…14). Both steps may actually occur in an asynchronous concerted step depending on the substrate (32,33). Note that a covalently bound inhibitor present in this structure has been omitted (34).…”