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Purification and characterization of extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus. Purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds
5'-Methylthioadenosine phosphorylase from Sulfolobus solfataricus, a thermoacidophilic archaeon optimally growing at 87 degrees C, has been purified to homogeneity. Reducing agents are not required for catalytic activity. The enzyme has a molecular mass of 160 kDa and is composed of six apparen...
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Published in: | The Journal of biological chemistry 1994-10, Vol.269 (40), p.24762-24769 |
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Main Authors: | , , , , |
Format: | Article |
Language: | English |
Subjects: | |
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Online Access: | Get full text |
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Summary: | 5'-Methylthioadenosine phosphorylase from Sulfolobus solfataricus, a thermoacidophilic archaeon optimally growing at 87 degrees
C, has been purified to homogeneity. Reducing agents are not required for catalytic activity. The enzyme has a molecular mass
of 160 kDa and is composed of six apparently identical subunits of 27 kDa. The NH2-terminal sequence shows high homology (50%)
with the NH2-terminal sequence of Escherichia coli purine nucleoside phosphorylase. Physicochemical and kinetic features are
reported. 5'-Methylthioadenosine phosphorylase is highly thermophilic, with an optimum temperature of 120 degrees C. The enzyme
is characterized by extreme thermal stability, remaining completely active after 2 h at 100 degrees C and showing half-inactivation
times of 15 and 5 min when incubated at 130 and 140 degrees C, respectively. An apparent melting temperature of 132 degrees
C has been calculated. After 24 h of incubation at room temperature no loss of activity is detected in the presence of 9 M
urea, 4 M guanidine hydrochloride, 0.075% SDS, 50% methanol, 50% ethanol, 50% dimethylformamide, 1 M NaCl, and 1% Triton X-100.
Data are also reported on the enzyme's resistance to proteolysis and on the effect of salts, detergents, solvents, and reducing
agents on enzyme thermostability. Labeling experiments with iodo[2-14C]acetic acid resulted in the incorporation of approximately
12 mol of labeled iodoacetate/mol of protein, indicating the presence of six disulfide bonds that, on the basis of SDS-polyacrylamide
gel electrophoresis, are probably positioned intersubunits, resulting in the organization of the enzyme into two trimers.
5'-Methylthioadenosine (MTA) phosphorylase is endowed with a broad substrate specificity, being able to phosphorolytically
cleave inosine, guanosine, and adenosine with a better efficiency than MTA, allowing us to hypothesize that in S. solfataricus
the same enzyme is responsible for the catabolism of MTA and of these purine nucleosides. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/s0021-9258(17)31457-6 |