Biochemical and structural characterization of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Pyrococcus horikoshii.

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.