Abstract

Adenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N⁶-adenosyl agarose. The enzyme shows a good stability to both temperature and pH. It appears to be a homohexamer of 164±5 kDa. Kinetic characterization confirmed the specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis (k_cat/K_m 2.1 × 10⁶ s⁻¹ M⁻¹), followed by 2′-deoxyadenosine (k_cat/K_m 4.2 × 10⁵ s⁻¹ M⁻¹). Apparently, the low specificity of adenosine phosphorylase towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding.

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