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 NH(3) or l-glutamine as the nitrogen source. The hydrolysis of glutamine is catalysed in the C-terminal glutamine amide transfer domain and the nascent NH(3) that is generated is transferred via an NH(3) tunnel [Endrizzi, J.A., Kim, H., Anderson, P.M. & Baldwin, E.P. (2004) Biochemistry43, 6447-6463] to the active site of the N-terminal 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, S.L. (2003) Biochem. J.369, 497-507]. To test our hypothesis that L109A CTP synthase has a constricted or a leaky NH(3) tunnel, we examined the ability of wild-type and L109A CTP synthases to utilize NH(3), NH(2)OH, and NH(2)NH(2) as exogenous substrates, and as nascent substrates generated via the hydrolysis of glutamine, gamma-glutamyl hydroxamate, and gamma-glutamyl hydrazide, respectively. We show that the uncoupling of the hydrolysis of gamma-glutamyl hydroxamate and nascent NH(2)OH production from N(4)-hydroxy-CTP formation is more pronounced with the L109A enzyme, relative to the wild-type CTP synthase. These results suggest that the NH(3) tunnel of L109A, in the presence of bound allosteric effector guanosine 5'-triphosphate, is not leaky but contains a constriction that discriminates between NH(3) and NH(2)OH on the basis of size.
Lunn
Faylene A
FA
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
Bearne
Stephen L
SL
D000641:Ammonia / Q000737:chemistry; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry*; D002384:Catalysis; D003570:Cytidine Triphosphate / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates / Q000737:chemistry; D005972:Glutaminase / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006877:Hydroxamic Acids / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013696:Temperature
D005971:Glutamates; D006877:Hydroxamic Acids; C001296:L-glutamic acid-gamma-hydrazide; C050614:glutamate-gamma-hydroxamic acid; D003570:Cytidine Triphosphate; D000641:Ammonia; D006160:Guanosine Triphosphate; D005972:Glutaminase; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D007930:Leucine