title: Thr-431 and Arg-433 are part of a conserved sequence motif of the glutamine amidotransferase domain of CTP synthases and are involved in GTP activation of the Lactococcus lactis enzyme.
abstract: A conserved sequence motif within the class 1 glutamine amidotransferase (GATase) domain of CTP synthases was identified. The sequence motif in the Lactococcus lactis enzyme is (429)GGTLRLG(435). This motif was present only in CTP synthases and not in other enzymes that harbor the GATase domain. Therefore, it was speculated that this sequence was involved in GTP activation of CTP synthase. Other members of the GATase protein family are not activated allosterically by GTP. Residues Thr-431 and Arg-433 were changed by site directed mutagenesis to the sterically similar residues valine and methionine, respectively. The resulting enzymes, T431V and R433M, had both lost the ability for GTP to activate the uncoupled glutaminase activity and showed reduced GTP activation of the glutamine-dependent CTP synthesis reaction. The T431V enzyme had a similar activation constant, K(A), for GTP, but the activation was only 2-3-fold compared with 35-fold for the wild type enzyme. The R433M enzyme was found to have a 10-15-fold lower K(A) for GTP and a concomitant decrease in V(app). The activation by GTP of this enzyme was about 7-fold. The kinetic parameters for saturation with ATP, UTP, and NH(4)Cl were similar for wild type and mutant enzymes, except that the R433M enzyme only had half the V(app) of the wild type enzyme when NH(4)Cl was the amino donor. The mutant enzymes T431V and R433M apparently had not lost the ability to bind GTP, but the signal transmitted through the enzyme to the active sites upon binding of the allosteric effector was clearly disrupted in the mutant enzymes.
authors.affiliation: Center for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark. martin@ccs.ki.ku.dk
authors.forename: Martin
authors.initials: M
authors.lastname: Willemoës
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D000878:Anthranilate Synthase; D001120:Arginine / Q000737:chemistry*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry*; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005972:Glutaminase / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology*; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019884:Nitrogenous Group Transferases / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry*; D014633:Valine / Q000737:chemistry
chemical_list: D000643:Ammonium Chloride; D013912:Threonine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D001120:Arginine; D008715:Methionine; D019884:Nitrogenous Group Transferases; D005972:Glutaminase; D000878:Anthranilate Synthase; C020655:anthranilate synthase, glutamine amidotransferase subunit; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D014633:Valine