Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity.

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress.

Recognition of the gluconeogenic enzyme, Pck1, via the Gid4 E3 ligase: An in silico perspective.

Gluconeogenesis, the reverse process of glycolysis, is a favorable mechanism at conditions of glucose deprivation. Pck1 is a rate-limiting gluconeogenic enzyme, where its deficiency or mutation contributes to serious clinical situations as neonatal hypoglycemia and liver failure. A recent report confirms that Pck1 is a target for proteasomal degradation through its proline residue at the penultimate position, recognized by Gid4 E3 ligase, but with a lack of informative structural details. In this study, we delineate the localized sequence motif, degron, that specifically interact with Gid4 ligase and unravel the binding mode of Pck1 to the Gid4 ligase by using molecular docking and molecular dynamics. The peptide/protein docking HPEPDOCK web server along with molecular dynamic simulations are applied to demonstrate the binding mode and interactions of a Pck1 wild type (SPSK) and mutant (K4V) with the recently solved structure of Gid4 ligase. Results unveil a distinct binding mode of the mutated peptide compared with the wild type despite having comparable binding affinities to Gid4. Moreover, the four-residue peptide is found insufficient for Gid4 binding, while the seven-residue peptide suffices for binding to Gid4. The amino acids S134, K135, and N137 in the loop L1 (between ß1 and ß2) of the Gid4 are essential for the stabilization of the seven-residue peptide in the binding site of the ligase. The presence of Val4 instead of Lys4 smashes the H-bonds that are formed between Lys4 and Gid4 in the wild type peptide, making the peptide prone to bind with the other side of the binding pocket (L4 loop of Gid4). The dynamics of Gid4 L3 loop is affected dramatically once K4V mutant Pck1 peptide is introduced. This opens the door to explore the mutation effects on the binding mode and smooth the path to target protein degradation by design competitive and non-competitive inhibitors.

Characterization of 3-[(Carboxymethyl)thio]picolinic Acid: A Novel Inhibitor of Phosphoenolpyruvate Carboxykinase.

Phosphoenolpyruvate carboxykinase (PEPCK) has traditionally been characterized for its role in the first committed step of gluconeogenesis. The current understanding of PEPCK's metabolic role has recently expanded to include it serving as a general mediator of tricarboxylic acid cycle flux. Selective inhibition of PEPCK in vivo and in vitro has been achieved with 3-mercaptopicolinic acid (MPA) (Ki ∼ 8 µM), whose mechanism of inhibition has been elucidated only recently. On the basis of crystallographic and mechanistic data of various inhibitors of PEPCK, MPA was used as the initial chemical scaffold to create a potentially more selective inhibitor, 3-[(carboxymethyl)thio]picolinic acid (CMP), which has been characterized both structurally and kinetically here. These data demonstrate that CMP acts as a competitive inhibitor at the OAA/PEP binding site, with its picolinic acid moiety coordinating directly with the M1 metal in the active site (Ki ∼ 29-55 µM). The extended carboxy tail occupies a secondary binding cleft that was previously shown could be occupied by sulfoacetate (Ki ∼ 82 µM) and for the first time demonstrates the simultaneous occupation of both OAA/PEP subsites by a single molecular structure. By occupying both the OAA/PEP binding subsites simultaneously, CMP and similar molecules can potentially be used as a starting point for the creation of additional selective inhibitors of PEPCK.

Phosphoenolpyruvate carboxykinase cytosolic and mitochondrial isoforms are expressed and active during hypoxia in the white shrimp Litopenaeus vannamei.

Hypoxic zones in marine environments are spreading around the world affecting the survival of many organisms. Marine animals have several strategies to respond to hypoxia, including the regulation of gluconeogenesis. Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of gluconeogenesis. The objective of this work was to study two isoforms of PEPCK, one mitochondrial (PEPKC-M) and one cytosolic (PEPCK-C), from the white shrimp Litopenaeus vannamei and the response to hypoxia. Both PEPCK isoforms are 72 kDa proteins and have 92% identity at the amino acid level. The mitochondrial isoform has a N-terminal signal peptide for mitochondrial import. Gene expression and enzymatic activity in subcellular fractions were detected in gills, hepatopancreas and muscle in normoxic and hypoxic conditions. Expression of PEPCK-C was higher than PEPCK-M in all the tissues and induced in response to hypoxia at 48 h in hepatopancreas, while the enzymatic activity of PEPCK-M was higher than PEPCK-C in gills and hepatopancreas, but not in muscle and also increased in response to hypoxia in hepatopancreas but decreased in gills and muscle. During limiting oxygen conditions, shrimp tissues obtain energy by inducing anaerobic glycolysis, and although gluconeogenesis implies energy investment, due to the need to maintain glucose homeostasis, these gluconeogenic enzymes are active with contrasting behaviors in the cytosol and mitochondrial cell compartments and appear to be up-regulated in hepatopancreas indicating this tissue pivotal role in gluconeogenesis during the response to hypoxia.

Asymmetric Anchoring Is Required for Efficient Ω-Loop Opening and Closing in Cytosolic Phosphoenolpyruvate Carboxykinase.

Mobile Ω-loops play essential roles in the function of many enzymes. Here we investigated the importance of a residue lying outside of the mobile Ω-loop element in the catalytic function of an H477R variant of cytosolic phosphoenolpyruvate carboxykinase using crystallographic, kinetic, and computational analysis. The crystallographic data suggest that the efficient transition of the Ω-loop to the closed conformation requires stabilization of the N-terminus of the loop through contacts between R461 and E588. In contrast, the C-terminal end of the Ω-loop undergoes changing interactions with the enzyme body through contacts between H477 at the C-terminus of the loop and E591 located on the enzyme body. Potential of mean force calculations demonstrated that altering the anchoring of the C-terminus of the Ω-loop via the H477R substitution results in the destabilization of the closed state of the Ω-loop by 3.4 kcal mol-1. The kinetic parameters for the enzyme were altered in an asymmetric fashion with the predominant effect being observed in the direction of oxaloacetate synthesis. This is exemplified by a reduction in kcat for the H477R mutant by an order of magnitude in the direction of OAA synthesis, while in the direction of PEP synthesis, it decreased by a factor of only 2. The data are consistent with a mechanism for loop conformational exchange between open and closed states in which a balance between fixed anchoring of the N-terminus of the Ω-loop and a flexible, unattached C-terminus drives the transition between a disordered (open) state and an ordered (closed) state.

Rosmarinic acid ameliorates hyperglycemia and insulin sensitivity in diabetic rats, potentially by modulating the expression of PEPCK and GLUT4.

BACKGROUND: Rosmarinic acid (RA) is a natural substance that may be useful for treating diabetes mellitus. The present study investigated the effects of RA on glucose homeostasis and insulin regulation in rats with streptozocin (STZ)-induced type 1 diabetes or high-fat diet (HFD)-induced type 2 diabetes. METHODS: Glucose homeostasis was determined using oral glucose tolerance tests and postprandial glucose tests, and insulin activity was evaluated using insulin tolerance tests and the homeostatic model assessment for insulin resistance. Additionally, the protein expression levels of PEPCK and GLUT4 were determined using Western blot analysis. RESULTS: RA administration exerted a marked hypoglycemic effect on STZ-induced diabetic rats and enhanced glucose utilization and insulin sensitivity in HFD-fed diabetic rats. These effects of RA were dose-dependent. Meanwhile, RA administration reversed the STZ- and HFD-induced increase in PEPCK expression in the liver and the STZ- and HFD-induced decrease in GLUT4 expression in skeletal muscle. CONCLUSION: RA reduces hyperglycemia and ameliorates insulin sensitivity by decreasing PEPCK expression and increasing GLUT4 expression.

Inhibition of Pig Phosphoenolpyruvate Carboxykinase Isoenzymes by 3-Mercaptopicolinic Acid and Novel Inhibitors.

There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 µM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 µM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C.

c.A2456C-substitution in Pck1 changes the enzyme kinetic and functional properties modifying fat distribution in pigs.

Cytosolic phosphoenolpyruvate carboxykinase, PCK1, is one of the main regulatory enzymes of gluconeogenesis and glyceroneogenesis. The substitution of a single amino acid (Met139Leu) in PCK1 as a consequence of a single nucleotide polymorphism (SNP), c.A2456C, is associated in the pig to a negative phenotype characterized by reduced intramuscular fat content, enhanced backfat thickness and lower meat quality. The p.139L enzyme shows reduced kcat values in the glyceroneogenic direction and enhanced ones in the anaplerotic direction. Accordingly, the expression of the p.139L isoform results in about 30% lower glucose and 9% lower lipid production in cell cultures. Moreover, the ability of this isoform to be acetylated is also compromised, what would increase its susceptibility to be degraded in vivo by the ubiquitin-proteasome system. The high frequency of the c.2456C allele in modern pig breeds implies that the benefits of including c.A2456C SNP in selection programs could be considerable.

Utilization of Substrate Intrinsic Binding Energy for Conformational Change and Catalytic Function in Phosphoenolpyruvate Carboxykinase.

Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Previous work has demonstrated that the enzyme cycles between a catalytically inactive open state and a catalytically active closed state. The transition of the enzyme between these states requires the transition of several active site loops to shift from mobile, disordered structural elements to stable ordered states. The mechanism by which these disorder-order transitions are coupled to the ligation state of the active site however is not fully understood. To further investigate the mechanisms by which the mobility of the active site loops is coupled to enzymatic function and the transitioning of the enzyme between the two conformational states, we have conducted structural and functional studies of point mutants of E89. E89 is a proposed key member of the interaction network of mobile elements as it resides in the R-loop region of the enzyme active site. These new data demonstrate the importance of the R-loop in coordinating interactions between substrates at the OAA/PEP binding site and the mobile R- and Ω-loop domains. In turn, the studies more generally demonstrate the mechanisms by which the intrinsic ligand binding energy can be utilized in catalysis to drive unfavorable conformational changes, changes that are subsequently required for both optimal catalytic activity and fidelity.

Inhibition and Allosteric Regulation of Monomeric Phosphoenolpyruvate Carboxykinase by 3-Mercaptopicolinic Acid.

For almost 40 years, it has been known that tryptophan metabolites and picolinic acid analogues act as inhibitors of gluconeogenesis. Early studies observed that 3-mercaptopicolinic acid (MPA) was a potent hypoglycemic agent via inhibition of glucose synthesis through the specific inhibition of phosphoenolpyruvate carboxykinase (PEPCK) in the gluconeogenesis pathway. Despite prior kinetic investigation, the mechanism of the inhibition by MPA is unclear. To clarify the mechanism of inhibition exerted by MPA on PEPCK, we have undertaken structural and kinetic studies. The kinetic data in concert with crystallographic structures of PEPCK in complex with MPA and the substrates for the reaction illustrate that PEPCK is inhibited by the binding of MPA at two discrete binding sites: one acting in a competitive fashion with PEP/OAA (∼10 µM) and the other acting at a previously unidentified allosteric site (Ki ∼ 150 µM). The structural studies suggest that binding of MPA to the allosteric pocket stabilizes an altered conformation of the nucleotide-binding site that in turn reduces the affinity of the enzyme for the nucleotide.

Dynamic behavior of rat phosphoenolpyruvate carboxykinase inhibitors: new mechanism for enzyme inhibition.

As an enzyme acting at the junction of gluconeogenic pathway, phosphoenolpyruvate carboxykinase (PEPCK) controls substrate flow from Krebs cycle toward glucose production. Therefore, it would be advantageous to design effective inhibitors to inactivate PEPCK in diabetes mellitus and other abnormalities caused by insulin resistance. Such inhibitors may compensate the metabolic consequences of ex-activity of PEPCK at these conditions. Understanding the mechanism by which inhibitors exert their effect on enzyme activity is of great interest for designing stronger inhibitors. In the present work, molecular dynamic simulations were used to study enzyme-inhibitor interactions. Our results indicate that inhibitors of PEPCK with their short chains interact with enzyme active site through non-covalent interactions of electrostatic and hydrogen bond nature. The data also show that inhibitors neither reach a stable state in their binding site nor make static complex with the enzyme active site. Instead, they interact with functional groups of active site residues in a dynamic fashion. In this way, oxalate and sulfoacetate carrying two negative groups of higher charge density and optimum spacing from each other, show more dynamic behavior (lower stability in their binding site) and more inhibitory effects than other inhibitors used (phosphonoformate, phosphoglycolate and 3-phosphonopropionate).

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

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.

Nature of protein-CO2 interactions as elucidated via molecular dynamics.

Rising global temperatures require innovative measures to reduce atmospheric concentrations of CO(2). The most successful carbon capture technology on Earth is the enzymatic capture of CO(2) and its sequestration in the form of glucose. Efforts to improve upon or mimic this naturally occurring process will require a rich understanding of protein-CO(2) interactions. Toward that end, extensive all-atom molecular dynamics (MD) simulations were performed on the CO(2)-utilizing enzyme phosphoenolpyruvate carboxykinase (PEPCK). Preliminary simulations were performed using implicit and explicit solvent models, which yielded similar results: arginine, lysine, tyrosine, and asparagine enhance the ability of a protein to bind carbon dioxide. Extensive explicit solvent simulations were performed for both wild-type PEPCK and five single-point PEPCK mutants, revealing three prevalent channels by which CO(2) enters (or exits) the active site cleft, as well as a fourth channel (observed only once), the existence of which can be rationalized in terms of the position of a key Arg residue. The strongest CO(2) binding sites in these simulations consist of appropriately positioned hydrogen bond donors and acceptors. Interactions between CO(2) and both Mn(2+) and Mg(2+) present in PEPCK are minimal due to the stable protein- and solvent-based coordination environments of these cations. His 232, suggested by X-ray crystallography as being a potential important CO(2) binding site, is indeed found to be particularly "CO(2)-philic" in these simulations. Finally, a recent mechanism, proposed on the basis of X-ray crystallography, for PEPCK active site lid closure is discussed in light of the MD trajectories. Overall, the results of this work will prove useful not only to scientists investigating PEPCK, but also to groups seeking to develop an environmentally benign, protein-based carbon capture, sequestration, and utilization system.

Activation of SIRT1 by resveratrol represses transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) by deacetylating hepatic nuclear factor 4alpha.

The SIRT1 activators isonicotinamide (IsoNAM), resveratrol, fisetin, and butein repressed transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C). An evolutionarily conserved binding site for hepatic nuclear factor (HNF) 4alpha (-272/-252) was identified, which was required for transcriptional repression of the PEPCK-C gene promoter caused by these compounds. This site contains an overlapping AP-1 binding site and is adjacent to the C/EBP binding element (-248/-234); the latter is necessary for hepatic transcription of PEPCK-C. AP-1 competed with HNF4alpha for binding to this site and also decreased HNF4alpha stimulation of transcription from the PEPCK-C gene promoter. Chromatin immunoprecipitation experiments demonstrated that HNF4alpha and AP-1, but not C/EBPbeta, reciprocally bound to this site prior to and after treating HepG2 cells with IsoNAM. IsoNAM treatment resulted in deacetylation of HNF4alpha, which decreased its binding affinity to the PEPCK-C gene promoter. In HNF4alpha-null Chinese hamster ovary cells, IsoNAM and resveratrol failed to repress transcription from the PEPCK-C gene promoter; overexpression of HNF4alpha in Chinese hamster ovary cells re-established transcriptional inhibition. Exogenous SIRT1 expression repressed transcription, whereas knockdown of SIRT1 by RNA interference reversed this effect. IsoNAM decreased the level of mRNA for PEPCK-C but had no effect on mRNA for glucose-6-phosphatase in AML12 mouse hepatocytes. We conclude that SIRT1 activation inhibited transcription of the gene for PEPCK-C in part by deacetylation of HNF4alpha. However, SIRT1 deacetylation of other key regulatory proteins that control PEPCK-C gene transcription also likely contributed to the inhibitory effect.

Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells.

Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni(2+)-NTA, and POROS Q columns obtained approximately 100mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M(r)=144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS-PAGE corresponding to the alpha (M(r) approximately 77,000) and beta (M(r) approximately 70,000) sGC subunits. It showed an A(430)/A(280)=1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k(cat)/K(m)=1.7 x 10(-3)s(-1)microM(-1) that increased to 5.8 x 10(-1)s(-1)microM(-1) upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure-function relationships.

Tyr235 of human cytosolic phosphoenolpyruvate carboxykinase influences catalysis through an anion-quadrupole interaction with phosphoenolpyruvate carboxylate.

Tyr235 of GTP-dependent phosphoenolpyruvate (PEP) carboxykinase is a fully invariant residue. The aromatic ring of this residue establishes an energetically favorable weak anion-quadrupole interaction with PEP carboxylate. The role of Tyr235 in catalysis was investigated via kinetic analysis of site-directed mutagenesis-derived variants. The Y235F change lowered the apparent K(m) for PEP by about six-fold, raised the apparent K(m) for Mn(2+) by about 70-fold, and decreased oxaloacetate (OAA)-forming activity by about 10-fold. These effects were due to an enhanced anion-quadrupole interaction between the aromatic side chain at position 235, which now lacked a hydroxyl group, and PEP carboxylate, which probably increased the distance between PEP and Mn(2+) and consequently affected the phosphoryl transfer step and overall catalysis. For the Y235A and Y235S changes, an elimination of the favorable edge-on interaction increased the apparent K(m) for PEP by four- and six-fold, respectively, and the apparent K(m) for Mn(2+) by eight- and six-fold, respectively. The pyruvate kinase-like activity, representing the PEP dephosphorylation step of the OAA-forming reaction, was affected by the substitutions in a similar way to the complete reaction. These observations indicate that the aromatic ring of Tyr235 helps to position PEP in the active site and the hydroxyl group allows an optimal PEP-Mn(2+) distance for efficient phosphoryl transfer and overall catalysis. The Y235A and Y235S changes drastically reduced the PEP-forming and OAA decarboxylase activities, probably due to the elimination of the stabilizing interaction between Tyr235 and the respective products, PEP and pyruvate.

Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection.

The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics and catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.

Structure of a GTP-dependent bacterial PEP-carboxykinase from Corynebacterium glutamicum.

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2(1) with four molecules per asymmetric unit. The 2.3A resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guanine binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.