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Journal of Bacteriology, July 2003, p. 4233-4242, Vol. 185, No. 14
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.14.4233-4242.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Mechanisms of Activation of Phosphoenolpyruvate Carboxykinase from Escherichia coli by Ca²⁺ and of Desensitization by Trypsin

Athena Sudom,¹ Robert Walters,² Landon Pastushok,² Douglas Goldie,² Lata Prasad,¹ Louis T. J. Delbaere,¹ and Hughes Goldie^2*

Departments of Biochemistry,¹ Microbiology and Immunology University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5²

Received 30 January 2003/ Accepted 24 April 2003

The 1.8-Å resolution structure of the ATP-Mg²⁺-Ca²⁺-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg²⁺-Mn²⁺-pyruvate-PCK, except for the Ca²⁺ and Mn²⁺ binding sites. Ca²⁺ 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 Ca²⁺ bound only at the active site, indicating that there is likely no surface allosteric site. ⁴⁵Ca²⁺ bound to PCK with a K_d of 85 µM and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca²⁺. Separate roles of Mg²⁺, which binds the nucleotide, and Ca²⁺, which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca²⁺-bound structure. Partial trypsin digestion abolishes Ca²⁺ 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 Ca²⁺ binding site, probably stabilizing the C terminus. Phe409Ala, Phe409, Phe413Ala, 397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca²⁺ 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.

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* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada. Phone: (306) 966-4312. Fax: (306) 966-4311. E-mail: goldie{at}duke.usask.ca.

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Journal of Bacteriology, July 2003, p. 4233-4242, Vol. 185, No. 14
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.14.4233-4242.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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