1OS1

Structure of Phosphoenolpyruvate Carboxykinase complexed with ATP,Mg, Ca and pyruvate.

Summary for 1OS1

• Entry DOI: 10.2210/pdb1os1/pdb
• Related: 1AQ2 1AYL 1OEN
• Descriptor: Phosphoenolpyruvate carboxykinase [ATP], MAGNESIUM ION, CALCIUM ION, ... (6 entities in total)
• Functional Keywords: enzyme mechanism, phosphotransfer, calcium, activation, lyase
• Biological source: Escherichia coli
• Cellular location: Cytoplasm: P22259
• Total number of polymer chains: 1
• Total formula weight: 60368.80

Authors

Sudom, A.,Walters, R.,Pastushok, L.,Goldie, D.,Prasad, L.,Delbaere, L.T.,Goldie, H. (deposition date: 2003-03-18, release date: 2003-09-23, Last modification date: 2023-11-15)

Primary citation

Sudom, A.,Walters, R.,Pastushok, L.,Goldie, D.,Prasad, L.,Delbaere, L.T.,Goldie, H.
Mechanisms of activation of phosphoenolpyruvate carboxykinase from Escherichia coli by Ca2+ and of desensitization by trypsin.
J.BACTERIOL., 185:4233-4242, 2003

PubMed Abstract: The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.

PubMed: 12837799
DOI: 10.1128/JB.185.14.4233-4242.2003

Experimental method

X-RAY DIFFRACTION (1.8 Å)