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Báo cáo khoa học: Aspartate transcarbamylase from the hyperthermophilic archaeonPyrococcus abyssi Insights into cooperative and allosteric mechanisms

Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi Insights into cooperative and allosteric mechanisms Sigrid Van Boxstael1, Dominique Maes2,3 and Raymond Cunin1 1 Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel, Belgium 2 Ultrastructuur, Vrije Universiteit Brussel, Belgium 3 Vlaams Interuniversitair Instituut voor Biotechnologie, Belgium Keywords allostery; aspartate transcarbamylase; cooperativity; inhibition by analogues; Pyrococcus abyssi Correspondence R. Cunin, Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium Fax: 32 2 629 1473 Tel: 32 2 629 1341 E-mail: rcunin@vub.ac.be (Received 15 December 2004, revised 1 March 2005, accepted 22 March 2005) doi:10.1111/j.1742-4658.2005.04678.x Aspartate transcarbamylase (ATCase) (EC 2.1.3.2) from the hyperthermophilic archaeon Pyrococcus abyssi was purified. | iFEBS Journal Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi Insights into cooperative and allosteric mechanisms Sigrid Van Boxstael1 Dominique Maes2 3 and Raymond Cunin1 1 Erfelijkheidsleer en Microbiologie Vrije Universiteit Brussel Belgium 2 Ultrastructuur Vrije Universiteit Brussel Belgium 3 Vlaams Interuniversitair Instituut voor Biotechnologie Belgium Keywords allostery aspartate transcarbamylase cooperativity inhibition by analogues Pyrococcus abyssi Correspondence R. Cunin Erfelijkheidsleer en Microbiologie Vrije Universiteit Brussel Pleinlaan 2 1050 Brussels Belgium Fax 32 2 629 1473 Tel 32 2 629 1341 E-mail rcunin@vub.ac.be Received 15 December 2004 revised 1 March 2005 accepted 22 March 2005 doi 10.1111 j.1742-4658.2005.04678.x Aspartate transcarbamylase ATCase EC 2.1.3.2 from the hyperthermo-philic archaeon Pyrococcus abyssi was purified from recombinant Escherichia coli cells. The enzyme has the molecular organization of class B microbial aspartate transcarbamylases whose prototype is the E. coli enzyme. P. abyssi ATCase is cooperative towards aspartate. Despite constraints imposed by adaptation to high temperature the transition between T- and R-states involves significant changes in the quaternary structure which were detected by analytical ultracentrifugation. The enzyme is allos-terically regulated by ATP activator and by CTP and UTP inhibitors . Nucleotide competition experiments showed that these effectors compete for the same sites. At least two regulatory properties distinguish P. abyssi ATCase from E. coli ATCase a UTP by itself is an inhibitor b whereas ATP and UTP act at millimolar concentrations CTP inhibits at micromolar concentrations suggesting that in P. abyssi inhibition by CTP is the major control of enzyme activity. While Vmax increased with temperature cooperative and allosteric effects were little or not affected showing that molecular adaptation to high temperature allows the flexibility required