Aspartate-107 and leucine-109 facilitate efficient coupling of glutamine hydrolysis to CTP synthesis by Escherichia coli CTP synthase

CTP synthase catalyses the ATP-dependent formation of CTP from UTP using either NH3 Or L-glutamine as the nitrogen source. GTP is required as an allosteric effector to promote glutamine hydrolysis. In an attempt to identify nucleotide-binding sites, scanning alanine mutagenesis was conducted on a highly conserved region of amino acid sequence (residues 102118) within the synthase domain of Escherichia coli CTP synthase. Mutant K102A CTP synthase exhibited wild-type activity with respect to NH, and glutamine; however, the R105A, 13107A, L109A and G110A enzymes exhibited wild-type NH3-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation. The E103A, R104A and H118A enzymes exhibited no glutamine-dependent activity and were only partially active with NH,. Although these observations were compatible with impaired activation by GTP, the apparent affinity of the D107A, L109A and G110A enzymes for GTP was reduced only 2-4-fold, suggesting that these residues do not play a significant role in GTP binding. In the presence of GTP, the k(cat) values for glutamine hydrolysis by the D107A and L109A enzymes were identical with that of wild-type CTP synthase. Overall, the kinetic properties of L109A CTP synthase were consistent with an uncoupling of glutamine hydrolysis from CTP formation that occurs because an NH3 tunnel has its normal structure altered or fails to form. L109F CTP synthase was prepared to block totally the putative NH3 tunnel; however, this enzyme's rate of glutamine-dependent CTP formation and glutaminase activity were both impaired. In addition, we observed that mutation of amino acids located between residues 102 and 118 in the synthase domain can affect the enzyme's glutaminase activity, suggesting that these residues interact with residues in the glutamine amide transfer domain because they are in close proximity or via a conformationally dependent signalling mechanism.