title: The importance of the strictly conserved, C-terminal glycine residue in phosphoenolpyruvate carboxylase for overall catalysis: mutagenesis and truncation of GLY-961 in the sorghum C4 leaf isoform.
abstract: Phosphoenolpyruvate carboxylase (PEPC) is a "multifaceted," allosteric enzyme involved in C4 acid metabolism in green plants/microalgae and prokaryotes. Before the elucidation of the three-dimensional structures of maize C4 leaf and Escherichia coli PEPC, our truncation analysis of the sorghum C4 homologue revealed important roles for the enzyme's C-terminal alpha-helix and its appended QNTG961 tetrapeptide in polypeptide stability and overall catalysis, respectively. Collectively, these functional and structural observations implicate the importance of the PEPC C-terminal tetrapeptide for both catalysis and negative allosteric regulation. We have now more finely dissected this element of PEPC structure-function by modification of the absolutely conserved C-terminal glycine of the sorghum C4 isoform by site-specific mutagenesis (G961(A/V/D)) and truncation (DeltaC1/C4). Although the C4 polypeptide failed to accumulate in a PEPC- strain (XH11) of E. coli transformed with the Asp mutant, the other variants were produced at wild-type levels. Although neither of these four mutants displayed an apparent destabilization of the purified PEPC homotetramer, all were compromised catalytically in vivo and in vitro. Functional complementation of XH11 cells under selective growth conditions was restricted progressively by the Ala, DeltaC1 and Val, and DeltaC4 modifications. Likewise, steady-state kinetic analysis of the purified mutant enzymes revealed corresponding negative trends in kcat and kcat/K0.5 (phosphoenolpyruvate) but not in K0.5 or the Hill coefficient. Homology modeling of these sorghum C-terminal variants against the structure of the closely related maize C4 isoform predicted perturbations in active-site molecular cavities and/or ion-pairing with essential, invariant Arg-638. These collective observations reveal that even a modest, neutral alteration of the PEPC C-terminal hydrogen atom side chain is detrimental to enzyme function.
authors.affiliation: Department of Biochemistry, Lincoln, Nebraska 68588-0664.
authors.forename: Wenxin
authors.initials: W
authors.lastname: Xu
authors.affiliation: Department of Biochemistry, Lincoln, Nebraska 68588-0664.
authors.forename: Shaheen
authors.initials: S
authors.lastname: Ahmed
authors.affiliation: Department of Chemistry, Lincoln, Nebraska 68588-0664.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Moriyama
authors.affiliation: Department of Biochemistry, Lincoln, Nebraska 68588-0664; Plant Science Initiative, University of Nebraska, Lincoln, Nebraska 68588-0664. Electronic address: rchollet1@unl.edu.
authors.forename: Raymond
authors.initials: R
authors.lastname: Chollet
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D005998:Glycine / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry* / Q000235:genetics*; D018515:Plant Leaves / Q000378:metabolism*; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D045868:Sorghum / Q000737:chemistry / Q000378:metabolism*; D003313:Zea mays / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D010730:Phosphoenolpyruvate Carboxylase; D005998:Glycine