Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus

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 (kcat/Km
2.1×106 s−1 M−1), followed by 2′-deoxyadenosine (kcat/Km 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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