Biochemical and structural characterization of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Pyrococcus horikoshii |
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Authors: | de Souza Dantas Deyse Ramos Dos Santos Camila Guimarães Pereira Gonçalo Amarante Medrano Francisco Javier |
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Institution: | Laboratorio de Gen?mica e Express?o, Departamento de Genética e Evolu??o, IB-UNICAMP, Caixa Postal 6109, CEP 13083-970, Campinas, Brazil. |
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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. |
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