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

Journal Article · Sun Jan 01 00:00:00 EST 2006 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M602299200· OSTI ID:930229

Xu, W; Ahmed, S; Moriyama, H; Chollet, R

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 QNTG{sup 961} 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 ({Delta}C1/C4). Although the C4 polypeptide failed to accumulate in a PEPC{sup -} 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, {Delta}C1 and Val, and {Delta}C4 modifications. Likewise, steady-state kinetic analysis of the purified mutant enzymes revealed corresponding negative trends in k{sub cat} and k{sub cat}/K0.5 (phosphoenolpyruvate) but not in K{sub 0.5} 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.

• Research Organization: Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: DE-AC02-98CH10886
• OSTI ID: 930229
• Report Number(s): BNL-80904-2008-JA; JBCHA3; TRN: US200822%%1408
• Journal Information: Journal of Biological Chemistry, Vol. 281; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

08 HYDROGEN
ATOMS
CARBOXYLASE
CATALYSIS
CAVITIES
DISTURBANCES
ELEMENTS
ENZYMES
ESCHERICHIA COLI
FUNCTIONALS
GLYCINE
GROWTH
HYDROGEN
IN VITRO
IN VIVO
KINETICS
LEVELS
MAIZE
METABOLISM
MODIFICATIONS
MUTAGENESIS
MUTANTS
PHOSPHOENOLPYRUVATE
POLYPEPTIDES
RESIDUES
SIMULATION
SORGHUM
STABILITY
STRAINS
national synchrotron light source