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

Wenxin Xu, Shaheen Ahmed, Hideaki Moriyama, Raymond Chollet

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 α-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 (ΔC1/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, ΔC1 and Val, and ΔC4 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.

Original languageEnglish (US)
Pages (from-to)17238-17245
Number of pages8
JournalJournal of Biological Chemistry
Volume281
Issue number25
DOIs
StatePublished - Jun 23 2006

Fingerprint

Phosphoenolpyruvate Carboxylase
Mutagenesis
Sorghum
Catalysis
Glycine
Protein Isoforms
Enzymes
Escherichia coli
Zea mays
Allosteric Regulation
Viridiplantae
Microalgae
Peptides
Phosphoenolpyruvate
Site-Directed Mutagenesis
Metabolism
Hydrogen
Catalytic Domain
Ions
Atoms

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{5f29348d1b084c8c87265ba836647053,
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 α-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 (ΔC1/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, ΔC1 and Val, and ΔC4 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.",
author = "Wenxin Xu and Shaheen Ahmed and Hideaki Moriyama and Raymond Chollet",
year = "2006",
month = "6",
day = "23",
doi = "10.1074/jbc.M602299200",
language = "English (US)",
volume = "281",
pages = "17238--17245",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "25",

}

TY - JOUR

T1 - The importance of the strictly conserved, C-terminal glycine residue in phosphoenolpyruvate carboxylase for overall catalysis

T2 - Mutagenesis and truncation of GLY-961 in the sorghum C4 leaf isoform

AU - Xu, Wenxin

AU - Ahmed, Shaheen

AU - Moriyama, Hideaki

AU - Chollet, Raymond

PY - 2006/6/23

Y1 - 2006/6/23

N2 - 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 α-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 (ΔC1/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, ΔC1 and Val, and ΔC4 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.

AB - 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 α-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 (ΔC1/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, ΔC1 and Val, and ΔC4 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.

UR - http://www.scopus.com/inward/record.url?scp=33745218323&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33745218323&partnerID=8YFLogxK

U2 - 10.1074/jbc.M602299200

DO - 10.1074/jbc.M602299200

M3 - Article

VL - 281

SP - 17238

EP - 17245

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 25

ER -