5'-METHYLTHIOADENOSINE PHOSPHORYLASE FROM CALDARIELLA-ACIDOPHILA - PURIFICATION AND PROPERTIES

The occurrence of 5′‐methylthioadenosine phosphorylase in Caldariella acidophila, a thermophilic bacterium growing optimally at 87°C, is reported. It represents the first example in prokaryotes of a phosphorolytic cleavage of the thioether. The reaction products, purified by ion‐exchange chromatography, have been identified as 5‐methylthioribose‐1‐phosphate and adenine by several analytical procedures. The enzyme has been purified to homogeneity in 32% yield by using DEAE‐cellulose and hydroxyapatite chromatography, gel filtration and isoelectric focusing. The enzyme shows a high degree of thermophilicity, its temperature optimum being at 93°C; furthermore no loss of activity is observable after exposure for 1 h at 100°C. The kinetic data indicate a sequential mechanism of the reaction. The apparent Km values are 0.095 mM for 5′‐methylthioadenosine and 6.1 mM for phosphate. The specificity of the reaction is rather strict. Experiments performed with analogues of the substrate, i.e. 5′‐methylthioinosine, 5′‐dimethylthioadenosine sulfonium salt, 5′‐n‐butylthioadenosine, 5′‐isobutylthioadenosine, 5′‐isobutylthioinosine, adenosylhomocysteine, 5′‐thioethanoladenosine, adenosine, indicate the relevance of the adenine amino group and the sulfur in thioether form in the binding to the enzyme protein. Copyright © 1979, Wiley Blackwell. All rights reserved