Ligand interactions with galactose oxidase: mechanistic insights

• Published in: Biophysical journal 1993-03, Vol.64 (3), p.762-772
• Main Authors: Whittaker, M.M., Whittaker, J.W.
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ALCOHOL DEHYDROGENASE; ALCOHOL DESHIDROGENASA; ALCOHOL OXIDOREDUCTASES; ALCOOL DESHYDROGENASE; Analytical, structural and metabolic biochemistry; ANION; ANIONES; ANIONS; azide; BINDING SITE; Binding Sites; Biological and medical sciences; Biophysical Phenomena; Biophysics; C.D; catalysis; Circular Dichroism; COBRE; COPPER; CUIVRE; E.S.R; ELECTRON PARAMAGNETIC RESONANCE SPECTROS; Electron Spin Resonance Spectroscopy; Enzyme Activation; Enzymes and enzyme inhibitors; ENZYMIC ACTIVITY; ESPECTROSCOPIA ESR; ESR SPECTROSCOPY; Fundamental and applied biological sciences. Psychology; galactose oxidase; Galactose Oxidase - chemistry; Galactose Oxidase - metabolism; HYPOMYCES; HYPOMYCES ROSELLUS; interaction; LIGAND; LIGANDOS; LIGANDS; mechanisms; Mitosporic Fungi - enzymology; Molecular Structure; NITRIC OXIDE; NITROGEN OXIDES; Oxidoreductases; OXIDOS DE NITROGENO; OXYDE D'AZOTE; Spectrophotometry; SPECTROSCOPIE RSE; Thermodynamics
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(93)81437-1

Interactions between galactose oxidase and small molecules have been explored using a combination of optical absorption, circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to detect complex formation and characterize the products. Anions bind directly to the cupric center in both active and inactive galactose oxidase, converting to complexes with optical and EPR spectra that are distinctly different from those of the starting aquo enzyme. Azide binding is coupled to stoichiometric proton uptake by the enzyme, reflecting the generation of a strong base (pKa > 9) in the active site anion adduct. At low temperature, the aquo enzyme converts to a form that exhibits the characteristic optical and EPR spectra of an anion complex, apparently reflecting deprotonation of the coordinated water. Anion binding results in a loss of the optical transition arising from coordinated tyrosine, implying displacement of the axial tyrosine ligand on forming the adduct. Nitric oxide binds to galactose oxidase, forming a specific complex exhibiting an unusual EPR spectrum with all g values below 2. The absence of Cu splitting in this spectrum and the observation that the cupric EPR signal from the active site metal ion is not significantly decreased in the complex suggest a nonmetal interaction site for NO in galactose oxidase. These results have been interpreted in terms of a mechanistic scheme where substrate binding displaces a tyrosinate ligand from the active site cupric ion, generating a base that may serve to deprotonate the coordinated hydroxyl group of the substrate, activating it for oxidation. The protein-NO interactions may probe a nonmetal O2 binding site in this enzyme.