Purification and Properties of Purine Nucleoside Phosphorylase fromBrevibacterium acetylicumATCC 954

Purine nucleoside phosphorylase of Brevibacterium acetylicum ATCC 954, which catalyzes the production of ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide), a potent antiviral agent, from purine nucleoside and 1,2,4-triazole-3-carboxamide in a high yield, was purified 49-fold. This enzyme had a molecular weight of 31,000 and was a monomer. The isoelectric point of the enzyme was 4.7. The optimal temperature and pH of inosine phosphorolyzing reaction catalyzed by the enzyme was around 8.5 and 70°C, respectively. The Michaelis constants for inosine, guanosine, and ribavirin were 1.43 mm, 2.44 mm and 2.08 mm, respectively, at 40°C. This enzyme appeared to be a SH enzyme because it was inactivated by SH reagents, p-chloromercuribenzoate and N-ethylmaleimide, and HgCl2. In addition, this enzyme was completely inactivated by AgNO3 and was slightly inhibited by CuSO4. It showed nucleoside-phosphorolyzing activity toward inosine, 2’-deoxyinosine, 2’,3’-dideoxyinosine, guanosine, 2’-deoxyguanosine, and xanthosine. However, adenosine and its derivatives could not be phosphorolyzed. This enzyme could not also phosphorolyze various 5’-mononucleotides. According to the amino terminal sequence analysis, the twenty residues from the amino terminal end of this enzyme were identified as follows: MTVNWNETRS-FLECKMQAKPE.