Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase (original) (raw)
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FEBS Letters, 1999
We have determined the crystal structure of Mn 2+bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X-ray diffraction at 2.6 A î resolution, and specified the location of enzyme-bound Mn 2+ , which is essential for catalytic activity. The electron density map reveals that Mn 2+ is bound to the side chain oxygens of Glu-506 and Asp-543, and located at the top of the K K/L L barrel in PEPC. The coordination sphere of Mn 2+ observed in E. coli PEPC is similar to that of Mn 2+ found in the pyruvate kinase structure. The model study of Mn 2+-bound PEPC complexed with phosphoenolpyruvate (PEP) reveals that the side chains of Arg-396, Arg-581 and Arg-713 could interact with PEP.
Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms
Archives of Biochemistry and Biophysics, 2003
Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) catalyzes the irreversible carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate and Pi using Mg 2þ or Mn 2þ as a cofactor. PEPC plays a key role in photosynthesis by C4 and Crassulacean acid metabolism plants, in addition to its many anaplerotic functions. Recently, three-dimensional structures of PEPC from Escherichia coli and the C4 plant maize (Zea mays) were elucidated by X-ray crystallographic analysis. These structures reveal an overall square arrangement of the four identical subunits, making up a ''dimer-of-dimers'' and an eight-stranded b barrel structure. At the Cterminal region of the b barrel, the Mn 2þ and a PEP analog interact with catalytically essential residues, confirmed by site-directed mutagenesis studies. At about 20 A A from the b barrel, an allosteric inhibitor (aspartate) was found to be tightly bound to downregulate the activity of the E. coli enzyme. In the case of maize C4-PEPC, the putative binding site for an allosteric activator (glucose 6-phosphate) was also revealed. Detailed comparison of the various structures of E. coli PEPC in its inactive state with maize PEPC in its active state shows that the relative orientations of the two subunits in the basal ''dimer'' are different, implicating an allosteric transition. Dynamic movements were observed for several loops due to the binding of either an allosteric inhibitor, a metal cofactor, a PEP analog, or a sulfate anion, indicating the functional significance of these mobile loops in catalysis and regulation. Information derived from these three-dimensional structures, combined with related biochemical studies, has established models for the reaction mechanism and allosteric regulation of this important C-fixing enzyme.
Science Access, 2001
The crystal structure of E.coli PEPC and Zea mays C4-form PEPC have been resolved recently, and we found an untraceable loop structure from K702 to G708 in E.coli numbering (from K762 to G768 in Zea mays numbering). This loop structure has flexibility and mobilty even in crystal state and participate in forming the active site (Kai et al. (1999) Proc. Natl. Acad. Sci. USA, 96, 823-828). Five mutants of this loop in Zea mays PEPC have been constructed. In the double mutation P765R/G766R, Vmax increased and extent of activation by an allosteric activator, G6P, increased. However, the Km value for Mg2+ increased. The two mutations P765R and P765G/G766R decreased the extent of activation by G6P. P765G mutant decreased the activity of the bicarbonate dependent PEP hydrolysis reaction and increased the extent of activation by G6P. In the insertional mutation of Arg residue between P765 and G766, Vmax increased and the extent of activation by G6P increased. These results indicated that thi...
ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK) is a key catabolic enzyme found in various species of bacteria, plants, and yeast. PEPCK may play a role in carbon fixation in aquatic ecosystems consisting of photosynthetic cyanobacteria. RuBisCO-based CO 2 fixation is prevalent in cyanobacteria through C 3 intermediates; however, a significant amount of carbon flows into C 4 acids during cyanobacterial photosynthesis. This indicates that a C 4 mechanism for inorganic carbon fixation is prevalent in cyanobacteria with PEPCK as an important -carboxylation enzyme. Newly available genomic information has confirmed the existence of putative PEPCK genes in a number of cyanobacterial species. This project represents the first structural and physicochemical study of cyanobacterial PEPCKs. Biocomputational analyses of cyanobacterial PEPCKs were performed and a homology model of Cyanothece sp. PCC 7424 PEPCK was generated. The modeled enzyme consists of an N-terminal and C-terminal domains with a mixed / topology with the active site located in a deep cleft between the two domains. Active site residues and those involved in metal ion coordination were found to be conserved in the cyanobacterial enzymes. An active site lid which is known to close upon substrate binding was also predicted. Amino acid stretches that are unique to cyanobacterial PEPCKs were also identified.
Biochimie, 2006
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn 2+ as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. Nature Struct. Biol. 4 (1997) 990-994] shows that Lys 213 is one of the ligands to Mn 2+ at the enzyme active site. Coordination of Mn 2+ to a lysyl residue is infrequent and suggests a low pK a value for the ε-NH 2 group of Lys 213 . In this work, we evaluate the role of neighboring Phe 416 in contributing to provide a low polarity microenvironment suitable to keep the ε-NH 2 of Lys 213 in the unprotonated form. Mutation Phe416Tyr shows that the introduction of a hydroxyl group in the lateral chain of the residue produces a substantial loss in the enzyme affinity for Mn 2+ , suggesting an increase of the pK a of Lys 213 . A study of the effect of pH on K m for Mn 2+ indicate that the affinity of recombinant wild type enzyme for the metal ion is dependent on deprotonation of a group with pK a of 7.1 ± 0.2, compatible with the low pK a expected for Lys 213 . This pK a value increases at least 1.5 pH units upon Phe416Tyr mutation, in agreement with the expected effect of an increase in the polarity of Lys 213 microenvironment. Theoretical calculations of the pK a of Lys 213 indicate a value of 6.5 ± 0.9, and it increases to 8.2 ± 1.6 upon Phe416Tyr mutation. Additionally, mutation Phe416Tyr causes a loss of 1.3 kcal mol −1 in the affinity of the enzyme for PEP, an effect perhaps related to the close proximity of Phe 416 to Arg 70 , a residue previously shown to be important for PEP binding.
Journal of Protein Chemistry, 2000
Molecular mechanics calculations have been employed to obtain models of the complexes between Saccharomyces cerevisiae phosphoenolpyruvate (PEP) kinase and the ATP analogs pyridoxal 5′-diphosphoadenosine (PLP-AMP) and pyridoxal 5′-triphosphoadenosine (PLP-ADP), using the crystalline coordinates of the ATP-pyruvate-Mn2+-Mg2+ complex of Escherichia coli PEP carboxykinase [Tari et al. (1997), Nature Struct. Biol. 4, 990–994]. In these models, the preferred conformation of the pyridoxyl moiety of PLP-ADP and PLP-AMP was established through rotational barrier and simulated annealing procedures. Distances from the carbonyl-C of each analog to ε-N of active-site lysyl residues were calculated for the most stable enzyme-analog complex conformation, and it was found that the closest ε-N is that from Lys290, thus predicting Schiff base formation between the corresponding carbonyl and amino groups. This prediction was experimentally verified through chemical modification of S. cerevisiae PEP carboxykinase with PLP-ADP and PLP-AMP. The results here described demonstrate the use of molecular modeling procedures when planning chemical modification of enzyme-active sites.
The Journal of Biochemistry, 1991
Phosphoenolpyruvate carboxylases (PEPC) [EC 4.1.1.31] from a wide variety of organisms contain a unique and highly conserved sequence, 578 FHGRGGSIGRGGAP 591 (coordinates for the Escherichia coli enzyme), which has been presumed to participate in the binding of phosphoenolpyruvate (PEP). Since previous chemical modification studies had suggested the importance of His for the catalytic activity, the role of His S7t was investigated by constructing variants of E. coli PEPC, in which this residue was substituted to Asn (H579N) or Pro (H579P). Kinetic studies with partially purified enzymes revealed the following: (1) The apparent maximal velocities in the presence of acetyl-CoA (CoASAc, one of the allosteric activators) were 29% and 5.4% of the wild-type enzyme, for H579N and H579P, respectively. (2) The half-saturation concentration for PEP was increased about 40-fold by the substitutions, while those for another substrate (HCO,~) and the metal cofactor (Mg 2+) were increased only 2-to 4-fold. (3) The half-saturation concentrations of four kinds of allosteric activators and of dioxane, an artificial activator, were also changed to various extents. Among them the most remarkable increase was observed for CoASAc (28-fold). (4) The concentration of an allosteric inhibitor, aspartate, required for 50% inhibition remained substantially unchanged. It was concluded that the imidazole group of His 57 " is not obligatory for the enzyme catalysis, but plays important roles in catalytic and regulatory functions. Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31] catalyzes the carboxylation of PEP to form oxaloacetate and inorganic phosphate. The enzyme is widespread in higher plants, algae, and many kinds of bacteria (1,2) and plays an anaplerotic role by replenishing C^dicarboxylic acids to the citric acid cycle (3). In C 4-and CAM plants, an alternate form of PEPC plays a key role in the initial CCvfixation (2). Moreover, the enzyme has many other physiological functions in higher plants (4). PEPCs from various sources are usually active as tetramers and their subunit molecular weights are about 100,000. Most of the PEPCs are allosteric in nature, but effector compounds differ widely depending on their sources. In the case of Eschericfua coli, the enzyme is regulated by four kinds of activators [CoASAc, fructose 1,6-bisphosphate (FBP), guanosine 3'-diphosphate 5'-diphosphate or GTP, and long-chain fatty acids] and by an inhibitor L-aspartate (5, 6). Furthermore, the enzyme is activated by several kinds of organic solvents such as dioxane and ethanol (7). The amino acid sequences of several PEPCs have been