Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus

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 N 6-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 × 10 6 s − 1 M − 1 ), followed by 2′-deoxyadenosine ( k cat/ K m 4.2 × 10 5 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.