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PDBsum entry 1kee

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Ligase PDB id
1kee
Jmol
Contents
Protein chains
1058 a.a. *
379 a.a. *
Ligands
ADP ×8
PO4 ×4
ORN ×4
NET ×4
Metals
_CL ×22
_MN ×12
__K ×32
Waters ×4176
* Residue conservation analysis

References listed in PDB file
Key reference
Title Inactivation of the amidotransferase activity of carbamoyl phosphate synthetase by the antibiotic acivicin.
Authors B.W.Miles, J.B.Thoden, H.M.Holden, F.M.Raushel.
Ref. J Biol Chem, 2002, 277, 4368-4373. [DOI no: 10.1074/jbc.M108582200]
PubMed id 11729189
Abstract
Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from 2 mol of ATP, bicarbonate, and glutamine. CPS was inactivated by the glutamine analog, acivicin. In the presence of ATP and bicarbonate the second-order rate constant for the inactivation of the glutamine-dependent activities was 4.0 x 10(4) m(-1) s(-1). In the absence of ATP and bicarbonate the second-order rate constant for inactivation of CPS was reduced by a factor of 200. The enzyme was protected against inactivation by the inclusion of glutamine in the reaction mixture. The ammonia-dependent activities were unaffected by the incubation of CPS with acivicin. These results are consistent with the covalent labeling of the glutamine-binding site located within the small amidotransferase subunit. The binding of ATP and bicarbonate to the large subunit of CPS must also induce a conformational change within the amidotransferase domain of the small subunit that enhances the nucleophilic character of the thiol group required for glutamine hydrolysis. The acivicin-inhibited enzyme was crystallized, and the three-dimensional structure was determined by x-ray diffraction techniques. The thiol group of Cys-269 was covalently attached to the dihydroisoxazole ring of acivicin with the displacement of a chloride ion.
Figure 1.
Fig. 1. Ribbon representation of an , -heterodimer of CPS. The small subunit is displayed in magenta. The large subunit is color-coded in green, yellow, blue, and red to indicate the carboxy phosphate, the oligomerization, the carbamoyl phosphate, and the allosteric subdomains, respectively. The tunnel connecting the three active sites is displayed in a wire mesh representation.
Figure 6.
Fig. 6. The binding of acivicin within the CPS small subunit. Electron density corresponding to the bound inhibitor is shown in A. The electron density map was contoured at 3 and calculated with coefficients of the form (F[o] F[c]), where F[o] was the native structure factor amplitude and F[c] was the calculated structure factor amplitude. Coordinates for the acivicin moiety and Cys-269 were not included in the Fourier synthesis. A schematic of the hydrogen bonding interactions between the inhibitor and the protein is given in B for comparison with that previously found for the binding of glutamine to the C269S mutant of CPS in C.
The above figures are reprinted by permission from the ASBMB: J Biol Chem (2002, 277, 4368-4373) copyright 2002.
PROCHECK
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