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

• DOI: 10.1042/bj20021110
• Publication Date: 2003-02-24T15:22:36Z
• Authors: Akshai IYENGAR; Stephen L. BEARNE
• Keywords: Glutaminase; Glutamine amidotransferase; Glutamate synthase

CTP synthase catalyses the ATP-dependent formation of from UTP using either NH(3) or L-glutamine as nitrogen source. GTP is required an allosteric effector to promote glutamine hydrolysis. In attempt identify nucleotide-binding sites, scanning alanine mutagenesis was conducted on a highly conserved region amino acid sequence (residues 102-118) within domain Escherichia coli synthase. Mutant K102A exhibited wild-type activity with respect and glutamine; however, R105A, D107A, L109A G110A enzymes NH(3)-dependent affinity for glutamine, but impaired glutamine-dependent formation. The E103A, R104A H118A no were only partially active NH(3). Although these observations compatible activation by GTP, apparent reduced 2-4-fold, suggesting that residues do not play significant role in binding. presence k (cat) values hydrolysis D107A identical Overall, kinetic properties consistent uncoupling occurs because tunnel has its normal structure altered fails form. L109F prepared block totally putative tunnel; this enzyme's rate glutaminase both impaired. addition, we observed mutation acids located between 102 118 can affect activity, interact amide transfer they are close proximity via conformationally dependent signalling mechanism.