Mechanisms of Activation of Phosphoenolpyruvate Carboxykinase from Escherichia coli by Ca 2+ and of Desensitization by Trypsin

Abstract

The 1.8-Å 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 μM 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, ΔPhe409, Phe413Ala, Δ397-521 (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.