Structural and biochemical evidence of the glucose 6-phosphate-allosteric site of maize C-4-phosphoenolpyruvate carboxylase: its importance in the overall enzyme kinetics
Por:
Munoz-Clares, Rosario A., Gonzalez-Segura, Lilian, Andres Juarez-Diaz, Javier, Mujica-Jimenez, Carlos
Publicada:
1 jun 2020
Resumen:
Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose
6-phosphate (G6P) and other phospho-sugars is of major physiological
relevance. Previous kinetic, site-directed mutagenesis and
crystallographic results are consistent with allosteric activation, but
the existence of a G6P-allosteric site was questioned and competitive
activation-in which G6P would bind to the active site eliciting the same
positive homotropic effect as the substrate phosphoenolpyruvate
(PEP)-was proposed. Here, we report the crystal structure of the
PEPC-C-4 isozyme from Zea mays with G6P well bound into the previously
proposed allosteric site, unambiguously confirming its existence. To
test its functionality, Asp239-which participates in a web of
interactions of the protein with G6P-was changed to alanine. The D239A
variant was not activated by G6P but, on the contrary, inhibited.
Inhibition was also observed in the wild-type enzyme at concentrations
of G6P higher than those producing activation, and probably arises from
G6P binding to the active site in competition with PEP. The lower
activity and cooperativity for the substrate PEP, lower activation by
glycine and diminished response to malate of the D239A variant suggest
that the heterotropic allosteric activation effects of free-PEP are also
abolished in this variant. Together, our findings are consistent with
both the existence of the G6P-allosteric site and its essentiality for
the activation of PEPC enzymes by phosphorylated compounds. Furthermore,
our findings suggest a central role of the G6P-allosteric site in the
overall kinetics of these enzymes even in the absence of G6P or other
phosphosugars, because of its involvement in activation by free-PEP.
Filiaciones:
Munoz-Clares, Rosario A.:
Univ Nacl Autonoma Mexico, Fac Quim, Dept Bioquim, Ciudad Univ, Mexico City 04510, DF, Mexico
Gonzalez-Segura, Lilian:
Univ Nacl Autonoma Mexico, Fac Quim, Dept Bioquim, Ciudad Univ, Mexico City 04510, DF, Mexico
Andres Juarez-Diaz, Javier:
Univ Nacl Autonoma Mexico, Fac Ciencias, Dept Biol Comparada, Ciudad Univ, Mexico City 04510, DF, Mexico
Mujica-Jimenez, Carlos:
Univ Nacl Autonoma Mexico, Fac Quim, Dept Bioquim, Ciudad Univ, Mexico City 04510, DF, Mexico
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