5'-Methylthioadenosine Phosphorylase from Caldariella acidophila. Purification and Properties

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Abstract
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.

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