TAILIEUCHUNG - Báo cáo khoa học: Limited proteolysis of Escherichia coli cytidine 5¢-triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis

Cytidine 5¢-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia orL-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed muta-genesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coliCTP synthase thatwere highly susceptible to proteolysis. . | Eur. J. Biochem. 270 2195-2206 2003 FEBS 2003 doi Limited proteolysis of Escherichia coli cytidine 5 -triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis Dave Simard Kerry A. Hewitt Faylene Lunn Akshai Iyengar and Stephen L. Bearne Department of Biochemistry and Molecular Biology Dalhousie University Halifax Nova Scotia Canada Cytidine 5 -triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the source of nitrogen. When glutamine is the substrate GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis Edman degradation and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues Lys187 Arg429 and Lys432 of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP UTP-binding site within the synthase domain and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 CTP ATP UTP GTP . The K187A mutant was resistant to proteolysis at this site could not catalyse CTP formation and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer GAT domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 1 and nucleotides did not protect these sites from cleavage. The R429A and R429A K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation. For these mutants the values of kcat Km and kcat for glutamine-dependent CTP formation were reduced 20-fold and 10-fold respectively relative to wild-type enzyme however the value of Km for .

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