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

AUTOR(ES)

Sudom, Athena

FONTE

American Society for Microbiology

RESUMO

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 Kd 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, ΔPhe409, Phe413Ala, Δ397-521 (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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