tailieunhanh - Báo cáo khoa học: Alternative substrates for wild-type and L109A E. coli CTP synthases Kinetic evidence for a constricted ammonia tunnel

Cytidine 5¢-triphosphate (CTP) synthase catalyses the ATP-dependent formation of CTP from uridine 5¢-triphosphate using either NH3orL-glutamine as the nitrogen source. The hydrolysis of glutamine is catalysed in the C-terminal glu-tamine amide transfer domain and the nascent NH3that is generated is transferred via an NH3 tunnel [Endrizzi, ., Kim, H., Anderson, . & Baldwin, . (2004) Biochemistry43, 6447–6463] to the active site of the N-ter-minal synthasedomainwhere theaminationreactionoccurs | Eur. J. Biochem. 271 4204-4212 2004 FEBS 2004 doi Alternative substrates for wild-type and L109A E. coli CTP synthases Kinetic evidence for a constricted ammonia tunnel Faylene A. Lunn and Stephen L. Bearne Department of Biochemistry and Molecular Biology Dalhousie University Halifax Nova Scotia Canada Cytidine 5 -triphosphate CTP synthase catalyses the ATP-dependent formation of CTP from uridine 5 -triphosphate using either NH3 or L-glutamine as the nitrogen source. The hydrolysis of glutamine is catalysed in the C-terminal glutamine amide transfer domain and the nascent NH3 that is generated is transferred via an NH3 tunnel Endrizzi . Kim H. Anderson . Baldwin . 2004 Biochemistry 43 6447-6463 to the active site of the N-ter-minal synthase domain where the amination reaction occurs. Replacement of Leu109 by alanine in Escherichia coli CTP synthase causes an uncoupling of glutamine hydrolysis and glutamine-dependent CTP formation Iyengar A. Bearne . 2003 Biochem. J. 369 497-507 . To test our hypothesis that L109A CTP synthase has a constricted or a leaky NH3 tunnel we examined the ability of wild-type and L109A CTP synthases to utilize NH3 NH2OH and NH2NH2 as exogenous substrates and as nascent substrates generated via the hydrolysis of glutamine y-glutamyl hydroxamate and y-glutamyl hydrazide respectively. We show that the uncoupling of the hydrolysis of y-glutamyl hydroxamate and nascent NH2OH production from N4-hydroxy-CTP formation is more pronounced with the L109A enzyme relative to the wild-type CTP synthase. These results suggest that the NH3 tunnel of L109A in the presence of bound allosteric effector guanosine 5 -triphosphate is not leaky but contains a constriction that discriminates between NH3 and NH2OH on the basis of size. Keywords amidotransferase ammonia tunnel CTP synthase glutaminase alternative substrates. Cytidine 5 -triphosphate CTP synthase CTPS EC UTP ammonia ligase ADP-forming catalyses .