tailieunhanh - Báo cáo khoa học: Allosteric properties of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermoacidophilic archaeon Sulfolobus solfataricus

Theuppgene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed inEscherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH when assayed at 60 C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. | iFEBS Journal Allosteric properties of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermoacidophilic archaeon Sulfolobus solfataricus Kaj F. Jensen1 Susan Arent2 Sine Larsen2 t and Lise Schack1 1 Department of BiologicalChemistry Institute of Molecular Biology University of Copenhagen Denmark 2 Centre for Crystallographic Studies Department of Chemistry University of Copenhagen Denmark Keywords extremophiles phosphoribosyl diphosphate pyrimidine nucleotide biosynthesis pyrimidine salvage thermostable enzymes Correspondence K. F. Jensen Department of Biological Chemistry Institute of Molecular Biology University of Copenhagen Solvgade 83H DK-1307 Copenhagen K Denmark Fax 45 3532204O Tel 45 35322020 E-mail kfj@ Present addresses Carlsberg Laboratory Department of Chemistry Gamle Carlsberg Vej10 DK-2500 Denmark tEuropean Synchrotron Radiation Facility ESRF-BP 220 F-38043 Grenoble Cedex France The upp gene encoding uracil phosphoribosyltransferase UPRTase from the thermoacidophilic archaeon Sulfolobus solfataricus was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH when assayed at 60 C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number kcat and raised the Km values for 5-phosphoribosyl-1-diphosphate PRPP and uracil by two- and 10-fold respectively. The inhibition by CTP was complex as it depended on the presence of the reaction product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors CTP and UMP can be bound simultaneously KD equal to 2 and pM respectively . The binding of each of the inhibitors was incompatible with binding of .