Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site

Dunten, Pete; Belunis, Charles; Crowther, R.; Hollfelder, Kurt; Kammlott, U.; Levin, Wayne; Michel, Hanspeter; Ramsey, Gwendolyn; Swain, Amy L.; Weber, David V.; Wertheimer, Stanley J.

doi:10.1006/jmbi.2001.5364

Cited by 72 publications

(108 citation statements)

References 24 publications

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“…Alignment of GTP-binding proteins shows that MnmE has the typical arrangement of GTP binding motifs, with G1 through G4 (1). This does not occur in the few GTPases that use GTP as energy source, such as adenylosuccinate synthetase or phosphoenolpyruvate carboxykinase (25,26,40). Our results indicate that the substitution of the invariant Asp of the G4 motif by an Asn residue changes specificity of MnmE from guanine to xanthine nucleotides, without affecting the mechanism of nucleoside triphosphate hydrolysis (Table II), as described for classical GTPases.…”

Section: Discussionmentioning

confidence: 53%

The GTPase Activity and C-terminal Cysteine of the Escherichia coli MnmE Protein Are Essential for Its tRNA Modifying Function

Yim¹,

Martínez-Vicente²,

Villarroya³

et al. 2003

Journal of Biological Chemistry

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The Escherichia coli MnmE protein is a three-domain protein that exhibits a very high intrinsic GTPase activity and low affinity for GTP and GDP. The middle GTPase domain, when isolated, conserves the high intrinsic GTPase activity of the entire protein, and the C-terminal domain contains the only cysteine residue present in the molecule. MnmE is an evolutionarily conserved protein that, in E. coli, has been shown to control the modification of the uridine at the wobble position of certain tRNAs. Here we examine the biochemical and functional consequences of altering amino acid residues within conserved motifs of the GTPase and C-terminal domains of MnmE. Our results indicate that both domains are essential for the MnmE tRNA modifying function, which requires effective hydrolysis of GTP. Thus, it is shown for the first time that a confirmed defect in the GTP hydrolase activity of MnmE results in the lack of its tRNA modifying function. Moreover, the mutational analysis of the GTPase domain indicates that MnmE is closer to classical GTPases than to GTP-specific metabolic enzymes. Therefore, we propose that MnmE uses a conformational change associated with GTP hydrolysis to promote the tRNA modification reaction, in which the C-terminal Cys may function as a catalytic residue. We demonstrate that point mutations abolishing the tRNA modifying function of MnmE confer synthetic lethality, which stresses the importance of this function in the mRNA decoding process.The evolutionarily conserved MnmE (TrmE) protein of Escherichia coli is a GTPase that differs extensively from regulatory GTPases such as p21 (1). Thus, MnmE exhibits a very high intrinsic GTPase hydrolysis rate and low affinity for GTP and GDP, and it can form self-assemblies. MnmE has a molecular mass of 50 kDa and is organized as a multidomain protein (see Fig. 1) consisting of an ϳ220-amino acid N-terminal domain, probably required for self-assembly, a middle GTPase domain, of about 160 residues, and an ϳ75-amino acid C-terminal domain, which contains the only Cys residue present in the protein. Strikingly, the isolated GTPase domain roughly conserves the guanine nucleotide binding and GTPase activities of the intact MnmE molecule (1).Null mnmE mutants are defective in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U 34 ) 1 (2), which is found in the wobble position (position 34) of tRNAs that read codons ending with A or G, in the mixed codon family boxes, specifically tRNAs for lysine and glutamic acid (3). The mnmA gene product (6) carries out thiolation in the 2-position of the wobble uridine (U 34 ), whereas mnmE controls the first step of the modification in the 5Ј-position, but it is unclear how many steps precede the formation of cmnm 5 s 2 U 34 (2, 4). Several data support that a second gene, named gidA, trmF, or mnmG, is also involved in the cmnm 5 group addition and that the MnmE activity precedes the activity of GidA (2, 5). The mnmC gene product has two enzymatic activities that transform the cm...

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Section: Discussionmentioning

confidence: 53%

The GTPase Activity and C-terminal Cysteine of the Escherichia coli MnmE Protein Are Essential for Its tRNA Modifying Function

Yim¹,

Martínez-Vicente²,

Villarroya³

et al. 2003

Journal of Biological Chemistry

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“…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)(3)(4)(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).…”

Section: From the Department Of Biochemistry And Molecular Biology Tmentioning

confidence: 93%

“…Mammalian PEPCK 2 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).…”

Section: From the Department Of Biochemistry And Molecular Biology Tmentioning

confidence: 99%

Structural Insights into the Mechanism of Phosphoenolpyruvate Carboxykinase Catalysis

Carlson

Holyoak

2009

Journal of Biological Chemistry

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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...

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“…It has been demonstrated previously in several enzyme systems that the motion of active site loop regions is either rate limiting or intimately coupled to the chemical step (39)(40)(41)(42). Previous structural studies have demonstrated that the predominant state for the active site lid domain in most complexes of PEPCK studied to date is the open/disordered state (28,29,32,35). On the basis of the structural studies of complexes mimicking the Michaelis and enolate intermediate states presented here, the thermodynamic favorability of the closed state appears to increase as the enzyme commits to catalysis by formation of the enolate intermediate.…”

Section: Induced Fit and Conformational Selectionmentioning

confidence: 99%

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

Sullivan

Holyoak

2008

Proc. Natl. Acad. Sci. U.S.A.

148

217

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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.enzyme dynamics ͉ population shift ͉ phosphoenolpyruvate carboxykinase I t has been 50 years since the original presentation of the induced fit hypothesis by Daniel Koshland (1) that built upon Emil Fisher's key-lock principle (2) and introduced the idea that the inherent dynamic properties of enzymes play an essential role in the processes of molecular recognition and catalysis. Over the last decade, with the advent of new instrumentation and novel experimental approaches there has been a resurgence in investigations focused upon understanding the specific ways in that these dynamic properties influence the ability of enzymes to achieve enormous levels of selectivity and catalytic power (refs. 3-6 and references therein).Presently there are two primary models used to explain the mechanism by which an enzyme can move toward adopting the final key-lock state ( Fig. 1): (i) the induced fit model and (ii) the conformational selection/population shift model (7-14). As defined originally by Koshland (1), the induced fit model states that the achievement of the final key-lock state, that is, the precise orientation of catalytic groups and residues necessary for catalysis, occurs only after changes in protein structure that are induced by the binding of a substrate. This pathway is represented by the equilibrium constants K 1 and K 2 in Fig. 1. Intrinsic to this model is the ability of the enzyme to form an ''encounter complex'' † (Fig. 1B). In this complex the enzyme is liganded identically to the eventual catalytically competent key-lock state; however, the conformational change leading to the active key-lock state has not yet occurred. In contrast, the conformational selection model states that the enzyme exists in multiple conformational states, of which the substrate has a high affinity for the active or key-lock state. In this model the ligand is suggested to bind to this lowly populated high-energy ...

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Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site

Cited by 72 publications

References 24 publications

The GTPase Activity and C-terminal Cysteine of the Escherichia coli MnmE Protein Are Essential for Its tRNA Modifying Function

The GTPase Activity and C-terminal Cysteine of the Escherichia coli MnmE Protein Are Essential for Its tRNA Modifying Function

Structural Insights into the Mechanism of Phosphoenolpyruvate Carboxykinase Catalysis

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

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