The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Recent studies have demonstrated that the enzyme contains a mobile active site lid domain that undergoes a transition between an open, disorded conformation and a closed, ordered conformation as the enzyme progresses through the catalytic cycle. The understanding of how this mobile domain functions in catalysis is incomplete. Previous studies showed that the closure of the lid domain stabilizes the reaction intermediate and protects the reactive intermediate from spurious protonation and thus contributes to the fidelity of the enzyme. To more fully investigate the roles of the lid domain in PEPCK function, we introduced three mutations that replaced the 11-residue lid domain with one, two, and three glycine residues. Kinetic analysis of the mutant enzymes demonstrates that none of the enzyme constructs exhibit any measurable kinetic activity, resulting in a decrease in the catalytic parameters of at least 10(6). Structural characterization of the mutants in complexes representing the catalytic cycle suggests that the inactivity is due to a role for the lid domain in the formation of the fully closed state of the enzyme that is required for catalytic function. In the absence of the lid domain, the enzyme is unable to achieve the fully closed state and is rendered inactive despite possessing all of the residues and substrates required for catalytic function. This work demonstrates how enzyme catalytic function can be abolished through the alteration of conformational equilibria despite all the elements required for chemical conversion of substrates to products remaining intact.

Figure 1

The active site loops of cPEPCK. The catalytically important mobile loop elements at the active site of PEPCK, the R-loop (85-92), the P-loop (284-292) and the Ω-loop lid (464-474) are labeled and colored blue, yellow and red, respectively. Oxalate and GTP are rendered as stick models and colored by atom type. The M1 and M2 manganese ions are colored as pink spheres. All graphics were generated using CCP4MG (15).

Figure 2

The domain structure of cPEPCK. The N-terminal lobe (residues 1-259), the nucleotide binding domain (residues 260-325 and 426-622), and the PEPCK-specific domain (residues 326-425), are rendered in red, blue and green, respectively. The molecule on the right is related to the molecule on the left by a rotation of 180° about the vertical axis illustrated. Oxalate and GTP are rendered as stick molecules colored by atom type and the manganese ions are rendered as pink spheres to illustrate the location of the bound substrates and the active site. The N- and C-termini are also labeled.

Figure 3

The amino acid sequence in the region of the PEPCK Ω-loop lid for WT and Ld_1g, _2g, and _3g PEPCK. The residues used to replace the Ω-loop in the deletion constructs are rendered in bold and underlined. The sequence of the WT Ω-loop lid is italicized and rendered in bold.

Figure 4

Active site regions of Ld _3g lidless PEPCK The A) PEPCK-βSP—Mn²⁺—Mn²⁺GTP , B) oxalate— Mn²⁺—Mn²⁺GTP, and the C)PEP— Mn²⁺—GDP complexes are illustrated. The active site residues are rendered as ball-and-stick models, the ligands are modeled as cylinders, and both are colored by atom type. The M1 and M2 Mn²⁺ ions are shown as pink spheres. The truncated Ω-loop lid is colored yellow. Dashed lines represent side chain-ligand interactions. 2Fo-Fc density for the ligands is shown as a blue mesh rendered at 1.5 σ. The dashed lines represent potential ligand-protein interactions.

Figure 5

Superposing of the Ω-loop lid regions of lidless PEPCK. The region of the structures corresponding to residues 450-496 is illustrated for the Ld_1g (green), Ld_2g (orange), Lg_3g (cyan), WT_open (grey) and WT_closed (black) PEPCK-Mn²⁺-ßSP-Mn²⁺GTP complexes. The residues R461 and H477 are labeled for reference.

Figure 6

Superposing of the open and closed enzyme forms of WT PEPCK and Ld_3g lidless PEPCK. The WT open (PGA-GDP, cyan) complex, the WT closed oxalate-GDP complex (gray), and Ld_3g lidless PEPCK in the oxalate-GTP complex (tan) are illustrated. The bound nucleotide in the WT-PGA-GDP complex is colored by atom type with tan carbons and the nucleotide in the closed WT-oxalate-GTP complex is colored by atom type with green carbons. This representation illustrates the impact of enzyme closure upon the position of the bound nucleotide. The large arrows illustrate the displacement in the C-terminal and PEPCK-specific domains progressing from the fully open, PGA-GDP complex, through the partially closed Ld_3g-oxalate-GTP complex, to the fully closed WT-oxalate-GTP complex. The orientation of the enzyme in this composite figure is the same as in Figure 2.

Figure 7

Alterations to the binding mode of GDP in the lidless A) Ld_1g, B) Ld_2g and C) Ld_3g PEPCK-Mn²⁺-PEP-Mn²⁺GDP complexes. The protein and nucleotide conformations corresponding to the normal position of GDP found in the WT PEPCK-Mn²⁺-PEP-Mn²⁺GDP complex are rendered in light blue. The protein and nucleotide conformation corresponding to the abnormal position of GDP found in the Ld_1g and Ld_2g-Mn²⁺-PEP-Mn²⁺GDP complexes are rendered in light green. In D) a superpositioning of the Ld_2g and Ld_3g complexes (panels B and C) is shown illustrating the changes in the position of the P-loop (delineated by S286 and T291) and the nucleotide binding pocket (234-443, 514-533) corresponding to the two modes of GDP binding.

Figure 8

The alternate conformation for the R-loop observed in the Ld_1g-oxalate-Mn²⁺-Mn²⁺GTP complex. The two conformations of the R-loop are shown in yellow (typical) and cyan (alternate conformation). Arginine 87 is rendered as a ball and stick model exhibiting a maximal displacement of 9.9Å at CZ (a). The other distances depicted are an ~6Å displacement at Cα of R87 (b) and the movement of Cα of residues A86(c) and V85 (d) by 5.5Å and 1.8Å, respectively. GTP (red), oxalate (tan) and the M1 manganese ion (pink) illustrate the location of the bound substrates and the position of the active site. Inset: the effect of R87 departure upon M1 metal ion coordination and oxalate interactions. Upon the movement of R87 out of the active site, K244 (green) moves from being a ligand to the M1 manganese ion to stabilizing the negative charge on oxalate (cyan). The coordination shell of the M1 manganese (pink) is filled upon departure of K244 by a water molecule (not shown).

Scheme 1

The reaction catalyzed by PEPCK.