Phosphoenolpyruvate carboxylase: Difference between revisions
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'''Phosphoenolpyruvate carboxylase''' (PEPC) catalyzes the irreversible β-carboxylation of PEP in the presence of HCO3- to yield OAA (oxaloacetate) and Pi ( inorganic phosphate), using Mg2 + as a cofactor <ref name="O'Leary2011">PMID: 21524275</ref>. It is a key element in the carbon fixation process in C4 plants <ref name="sage2004">PMID: 33873498</ref>. | '''Phosphoenolpyruvate carboxylase''' (PEPC) catalyzes the irreversible β-carboxylation of PEP in the presence of HCO3- to yield OAA (oxaloacetate) and Pi ( inorganic phosphate), using Mg2 + as a cofactor <ref name="O'Leary2011">PMID: 21524275</ref>. It is a key element in the carbon fixation process in C4 plants <ref name="sage2004">PMID: 33873498</ref>. | ||
== Overview == | == Overview == | ||
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== PEPC enzyme structure == | == PEPC enzyme structure == | ||
Overall, plant PEPC’s present a similar structure characterized by four 105-110kDa identical subunits with a conserved N-terminal serine-phosphorylation domain, forming homotetramers <ref name="kai2003"/><ref name="O'Leary2011"/>. X-ray crystallography studies on Escherichia coli and maize (Zea mays) PEPC’s show that the tetramer is comprised of two pairs of monomers with a greater amount of intersubunit contacts, suggesting a homotetrameric structure of a ‘‘dimer-of-dimers’’. The monomeric structure maize’s C4-PEPC monomer consists of an eight-stranded β barrel and 42 α helices <ref name="kai2003"/><ref name="Izui2004">PMID: 15725057</ref>. | Overall, plant PEPC’s present a similar structure characterized by four 105-110kDa identical subunits with a conserved N-terminal serine-phosphorylation domain, forming homotetramers <ref name="kai2003"/><ref name="O'Leary2011"/>. X-ray crystallography studies on Escherichia coli and maize (Zea mays) PEPC’s show that the tetramer is comprised of two pairs of monomers with a greater amount of intersubunit contacts, suggesting a homotetrameric structure of a ‘‘dimer-of-dimers’’. The monomeric structure maize’s C4-PEPC monomer consists of an eight-stranded β barrel and 42 α helices <ref name="kai2003"/><ref name="Izui2004">PMID: 15725057</ref>. The <scene name='57/573979/Cv/2'>active site</scene> of each monomer, bound to PEP and the <scene name='57/573979/Cv/3'>Mn+2 ion</scene> cofactor, is located in the C-terminus region of the β barrel <ref name="matsumura2002">/. | ||
The comparison between closely related C3 and C4 PEPC’s from F. pringlei (C3) and T. trinervia (C4) was very important in determining the changes responsible for the differences in the enzymes activity in C3 and C4 plants. Structural superposition of these two isoforms shows high levels of structural similarity, supported by the low backbone root-mean square deviation of 0.4 Å <ref name="Paulus2013"/>. Two important site-specific differences between the two structures are a substitution of the 884 residue located close to the feedback inhibitor-binding site, and another residue substitution at the 774 position. The first is largely responsible for the drastic differences observed between the inhibitor tolerances of the C3 and C4 PEPC’s, while the second is a key determinant for the different kinetic properties of F. pringlei and T. trinervia PEPC’s <ref name="blasing2000"/><ref name="Paulus2013"/>. | The comparison between closely related C3 and C4 PEPC’s from F. pringlei (C3) and T. trinervia (C4) was very important in determining the changes responsible for the differences in the enzymes activity in C3 and C4 plants. Structural superposition of these two isoforms shows high levels of structural similarity, supported by the low backbone root-mean square deviation of 0.4 Å <ref name="Paulus2013"/>. Two important site-specific differences between the two structures are a substitution of the 884 residue located close to the feedback inhibitor-binding site, and another residue substitution at the 774 position. The first is largely responsible for the drastic differences observed between the inhibitor tolerances of the C3 and C4 PEPC’s, while the second is a key determinant for the different kinetic properties of F. pringlei and T. trinervia PEPC’s <ref name="blasing2000"/><ref name="Paulus2013"/>. | ||
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== Allosteric regulation and reaction mechanism == | == Allosteric regulation and reaction mechanism == | ||
PEPC’s carboxylase activity is regulated by different post-translational mechanisms. In C4 and CAM plants, the phosphorylation of a serine residue near the N terminus (S15 in maize C4-PEPC) activates the enzyme by decreasing its sensitivity to allosteric inhibitors such as aspartate and malate and increasing activation by the positive allosteric regulator glucose 6-phosphate <ref name="O'Leary2011"/>. Studies on F. pringlei and T. trinervia, have positively identified the residues Arg641, Lys829, Arg888 and Asn964 as binding motif of the negative allosteric inhibitors aspartate and malate. In C3 PEPC, Arg884 provides an additional hydrogen bond for inhibitor binding, whereas in C4 PEPC isoforms the substitution of this residue by a glycine, reducing the enzymes sensitivity towards both feedback inhibitors <ref>PMID: 21491491</ref><ref name="Paulus2013"/>. The positive allosteric effector glucose 6-phosphate’s binding site has also been identified in the C4-PEPC of maize. X-ray crystallography of maize’s C4-PEPC in complex with sulfate ion (a positive effector analog of glucose 6-phosphate) revealed that the positive effector was bound to the enzyme at the dimer interface and was surrounded by four positively charged residues (R183, R184, R231, and R372 in the adjacent subunit <ref name="kai2003"/>. | PEPC’s carboxylase activity is regulated by different post-translational mechanisms. In C4 and CAM plants, the phosphorylation of a serine residue near the N terminus (S15 in maize C4-PEPC) activates the enzyme by decreasing its sensitivity to allosteric inhibitors such as aspartate and malate and increasing activation by the positive allosteric regulator glucose 6-phosphate <ref name="O'Leary2011"/>. Studies on F. pringlei and T. trinervia, have positively identified the residues Arg641, Lys829, Arg888 and Asn964 as binding motif of the negative allosteric inhibitors aspartate and malate <ref name="Paulus2013"/>. Similar studies have also identified the <scene name='57/573979/Cv/5'>aspartate binding site</scene> site in maize <ref name="matsumura2002"/>. In C3 PEPC, Arg884 provides an additional hydrogen bond for inhibitor binding, whereas in C4 PEPC isoforms the substitution of this residue by a glycine, reducing the enzymes sensitivity towards both feedback inhibitors <ref>PMID: 21491491</ref><ref name="Paulus2013"/>. The positive allosteric effector glucose 6-phosphate’s binding site has also been identified in the C4-PEPC of maize. X-ray crystallography of maize’s C4-PEPC in complex with sulfate ion (a positive effector analog of glucose 6-phosphate) revealed that the positive effector was bound to the enzyme at the dimer interface and was surrounded by four positively charged residues (R183, R184, R231, and R372 in the adjacent subunit <ref name="kai2003"/>. | ||
Most proposed catalytic reaction mechanisms suggest that PEPC catalyzes an ordered multistep reaction in which the preferred order of reactant binding to the active site are: first the bivalent cation ( Mg2+ or Mn2+), then PEP and lastly bicarbonate (HCO3- ) <ref name="kai2003"/><ref name="Izui2004"/>. In a review article on the subject, Izui et al., 2004 <ref name="Izui2004"/>, proposes a detailed reaction mechanism for the enzyme, based on maize and E.coli PEPC structures in which PEP is located in a hydrophobic pocket consisting of W248, L504, and M538 residues. In this model, H177 is a critically important catalytic base as it is supposed to play a role in stabilizing the carboxyphosphate intermediate and abstracting a proton from its carboxyl group. | Most proposed catalytic reaction mechanisms suggest that PEPC catalyzes an ordered multistep reaction in which the preferred order of reactant binding to the active site are: first the bivalent cation ( Mg2+ or Mn2+), then PEP and lastly bicarbonate (HCO3- ) <ref name="kai2003"/><ref name="Izui2004"/>. In a review article on the subject, Izui et al., 2004 <ref name="Izui2004"/>, proposes a detailed reaction mechanism for the enzyme, based on maize and E.coli PEPC structures in which PEP is located in a hydrophobic pocket consisting of W248, L504, and M538 residues. In this model, H177 is a critically important catalytic base as it is supposed to play a role in stabilizing the carboxyphosphate intermediate and abstracting a proton from its carboxyl group. | ||