Abstract
The 6-oxopurine phosphoribosyltransferase (HPRT, EC 2.4.2.8) from the hyperthermophile Pyrococcus horikoshii was expressed in Escherichia coli and purified. Steady-state kinetic studies indicated that the enzyme is able to use hypoxanthine, guanine and xanthine. The first two substrates showed similar catalytic efficiencies, and xanthine presented a much lower value (around 20 times lower), but the catalytic constant was comparable to that of hypoxanthine. The enzyme was not able to bind to GMP-agarose, but was able to bind the other reverse reaction substrate, inorganic pyrophosphate, with low affinity (K(d) of 4.7+/-0.1 mM). Dynamic light scattering and analytical gel filtration suggested that the enzyme exists as a homohexamer in solution.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Archaeal Proteins / chemistry
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Archaeal Proteins / genetics
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Archaeal Proteins / metabolism*
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Chromatography, Gel
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Circular Dichroism
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Dimerization
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Electrophoresis, Polyacrylamide Gel
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Guanine / metabolism
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Guanosine Monophosphate / metabolism
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Hypoxanthine / metabolism
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Molecular Sequence Data
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Pentosyltransferases / chemistry
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Pentosyltransferases / genetics
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Pentosyltransferases / metabolism*
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Pyrococcus horikoshii / enzymology*
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Pyrococcus horikoshii / genetics
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Sequence Homology, Amino Acid
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Xanthine / metabolism
Substances
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Archaeal Proteins
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Xanthine
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Hypoxanthine
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Guanine
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Guanosine Monophosphate
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Pentosyltransferases
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hypoxanthine-guanine-xanthine phosphoribosyltransferase