Structural Insights into the Mechanism of Phosphoenolpyruvate Carboxykinase Catalysis

Abstract

Mammalian PEPCK2 catalyzes the reversible formation of PEP from OAA and GTP (or ITP) in a divalent cation-dependent reaction (Scheme 1), as was elegantly discussed in the first minireview of this series on PEPCK (1).In this third minireview, high-resolution crystal structures of mammalian PEPCK are examined to gain insights into the mechanism of PEPCK catalysis, including the reaction's reversibility and nucleotide specificity. Regarding reaction reversibility, PEPCK is responsible for regeneration of the high-energy phosphoryl donor PEP from the unstable, activated ␤-keto acid OAA. When coupled with pyruvate carboxylase, PEPCK reverses the essentially irreversible formation of pyruvate and ATP from PEP and ADP in the glycolytic reaction catalyzed by pyruvate kinase. As illustrated (Fig. 1), PEPCK could achieve this feat by stabilizing the inherently unstable enolate form of pyruvate generated by decarboxylation of OAA (Scheme 1). Stabilization of this intermediate would reduce the energetic cost for phosphoryl transfer by ϳ30 kJ mol Ϫ1 relative to direct reversal of the pyruvate kinase-catalyzed reaction. An energetic driving force for the pyruvate kinase reaction is the favorable tautomerization of the high-energy enol to its corresponding keto form; in contrast, by stabilizing the enolate, PEPCK could prevent its energetically favorable protonation and tautomerization, allowing phosphoryl transfer to occur. Thus, by stabilizing this intermediate in a high-energy state, the PEPCK reaction would be energetically rendered freely reversible; the crystal structures that will be described indicate that PEPCK does, in fact, stabilize the enolate intermediate.The recent structures of PEPCK from human, rat, and chicken (2-5), the enzymes from Trypanosoma cruzi (6), Anaerobiospirillum succiniciproducens (7), and Corynebacterium glutamicum (8), and earlier work on the isozyme from Escherichia coli (9 -13) illustrate that the active-site residues and architecture are well conserved, despite what is rather poor overall sequence homology when comparing members of the ATP-and GTP-dependent families.3 As detailed in this minireview, the cationic environment of the active site, dominated by the juxtaposition of two divalent metal ions and the positioning of lysine and arginine residues, is well suited to allow for the stabilization of the enolate intermediate discussed above and to facilitate phosphoryl transfer.An informative aspect of the PEPCK-catalyzed reaction revealed by the recent structural data on the GTP-dependent isozyme from rat is the illumination of the previously unappreciated role of conformational changes occurring in the active site during the catalytic cycle (5). The most prevalent mobile feature illustrated by the structural work is a 10-residue ⍀-loop lid domain whose closure is potentially capable of protecting the enolate intermediate (Fig. 2) (2-5). A similar domain is present in ATPdependent PEPCK, as represented by the E. coli enzyme, which was the first PEPCK to be structurally characterized (9). Th...

Increasing the Conformational Entropy of the Ω-Loop Lid Domain in Phosphoenolpyruvate Carboxykinase Impairs Catalysis and Decreases Catalytic Fidelity,

“…In general, our model of lid dynamics in PEPCK proposes that not all of the free energy from substrate association goes into forming a tight binding E-S complex, but that some of that energy is partitioned to the protein. This input energy modifies the free energy landscape that defines the conformation of the protein, and results in the closed lid state becoming more thermodynamically favorable as substrates associate (6, 9). This general idea has been presented previously (41, 42).…”

“…Due to the inherent reactivity of the enolate intermediate, primarily its energetically favorable protonation resulting in pyruvate, we have suggested that the protection/stabilization of the enolate by the enzyme is key to the reversible nature of the PEPCK catalyzed reaction (Scheme 1) (6). …”

The Ω-Loop Lid Domain of Phosphoenolpyruvate Carboxykinase Is Essential for Catalytic Function

“…Specifically, inspection of the structural data reveals several mobile loops whose conformation and/or mobility is impacted by the ligation state of the enzyme along the reaction coordinate. The most noticeable elements are the R-loop (residues 85-92), the P-loop (residues 284-292), and the Ω-loop lid (residues 464-474) (Figure 1)(20). Further, similar to what is observed in the ATP-dependent isoform from E.coli (21) a global motion that involves the opening and closing of the N- and C-terminal lobes of the enzyme is also observed (20).…”

“…The most noticeable elements are the R-loop (residues 85-92), the P-loop (residues 284-292), and the Ω-loop lid (residues 464-474) (Figure 1)(20). Further, similar to what is observed in the ATP-dependent isoform from E.coli (21) a global motion that involves the opening and closing of the N- and C-terminal lobes of the enzyme is also observed (20). Due to their location at the active site, the R-loop and the P-loop are thought to be involved directly in substrate binding and catalysis.…”

“…First, its closure/ordering upon nucleotide binding removes a potential steric clash between T465 on the Ω-loop and A287 on the P-loop allowing the Ω-loop lid to sample its closed conformation. Secondly, typical to other kinases it cradles the β- and γ-phosphates of GDP/GTP and positions the invariant P-loop lysine (K290 in cPEPCK) in a position to activate the phosphate leaving group allowing for direct inline phosphoryl transfer (20). Once GTP is positioned for phosphoryl transfer and OAA is bound, the structural data suggest that the chemical reaction is initiated by the movement of the N- and C-terminal lobes toward one another via a ‘clam like’ closure of the two domains.…”