Stereochemistry of the carboxylation reaction catalyzed by the ATP-dependent phosphoenolpyruvate carboxykinases from Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens (original) (raw)
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Biochemistry, 1992
The catalytic mechanism of phosphoenolpyruvate (PEP) carboxylase from Zea mays has been studied using (2)and (E)-3-fluorophosphoenolpyruvate (F-PEP) as substrates. Both (2)and (E)-F-PEP partition between carboxylation to produce 3-fluorooxalacetate and hydrolysis to produce 3-fluoropyruvate. Carboxylation accounts for 3% of the reaction observed with (Z)-F-PEP, resulting in the formation of (R)-3-fluorooxalacetate, and for 86% of the reaction of (E)-F-PEP forming (S)-3-fluorooxalacetate. Carboxylation of F-PEP occurs on the 2-re face, which corresponds to the 2-si face of PEP. The partitioning of F-PEP between carboxylation and hydrolysis is insensitive to pH but varies with metal ion. Use of '*O-labeled bicarbonate produces phosphate that is multiply labeled with l80; in addition, I8O is also incorporated into residual (2)and (E)-F-PEP. The 13(V / K) isotope effect on the carboxylation of F-PEP catalyzed by PEP carboxylase at pH 8.0, 25 OC, is 1.049 f 0.003 for (Z)-F-PEP and 1.009 i 0.006 for (E)-F-PEP. These results are consistent with a mechanism in which carboxylation of PEP occurs via attack of the enolate of pyruvate on C 0 2 rather than carboxy phosphate. In this mechanism phosphorylation of bicarbonate to give carboxy phosphate and decarboxylation of the latter are reversible steps. An irreversible step, however, precedes partitioning between carboxylation to give oxalacetate and release of C02, which results in hydrolysis of PEP. O n e of the most effective tools for studying enzyme mechanisms is the use of alternate substrates, among which fluorinated analogues have been widely used (Abeles & Alston, 1990). The fluorinated analogue of PEP, 3-fluorophosphoenolpyruvate (F-PEP),' has been successfully employed in studying the mechanisms of a variety of PEP-utilizing enzymes. F-PEP has been shown to be a substrate for pyruvate kinase (Stubbe & Kenyon, 1972; Duffy & Nowak, 1984), enolase (Stubbe & Kenyon, 1972; Duffy & Nowak, 1984), phosphoenolpyruvate carboxykinase (Duffy & Nowak, 1984), and pyruvate phosphate dikinase (Duffy & Nowak, 1984). In addition, F-PEP has been used to determine the stereochemistry of the reaction catalyzed by phosphoenolpyruvate carboxykinase (Hwang & Nowak, 1986). Recently, two independent studies of the substrate activity of F-PEP with maize PEP carboxylase have been reported (Diaz et al., 1988; Gonzalez & Andreo, 1988). Both investigations demonstrated that the primary reaction of F-PEP with PEP carboxylase was hydrolysis, resulting in the formation of 3-fluoropyruvate. Diaz et al. (1988) reported that 14% of F-PEP was carboxylated to form 3-fluorooxalacetate (F-OM). Gonzalez and Andreo
Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: relevance of arginine 70 for catalysis
Biochimie, 2004
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase is a key enzyme of the gluconeogenic pathway and catalyzes the decarboxylation of oxaloacetate and transfer of the c-phosphoryl group of ATP to yield PEP, ADP, and CO 2 in the presence of a divalent metal ion. Previous experiments indicate that mutation of amino acid residues at metal site 1 decrease the enzyme catalytic efficiency and the affinity of the protein for PEP, evidencing the relevance of hydrogen-bond interactions between PEP and water molecules of the first coordination sphere of the metal ion for catalysis [Biochemistry 41 (2002) 12763]. To further understand the function of amino acid residues located in the PEP binding site, we have now addressed the catalytic importance of Arg 70 , whose guanidinium group is close to the PEP carboxyl group. Arg 70 mutants of PEP carboxykinase were prepared, and almost unaltered kinetic parameters were found for the Arg 70 Lys PEP carboxykinase, while a decrease in 4-5 orders of magnitude for the catalytic efficiency was detected for the Arg70Gln and Arg70Met altered enzymes. To evaluate the enzyme interaction with PEP, the phosphopyridoxyl-derivatives of wild type, Arg70Lys, Arg70Gln, and Arg70Met S. cerevisiae PEP carboxykinase were prepared, and the change in the fluorescence emission of the probe upon PEP binding was used to obtain the dissociation equilibrium constant of the corresponding derivatized enzyme-PEP-Mn 2+ complex. The titration experiments showed that a loss in 2.1 kcal/mol in PEP binding affinity is produced in the Arg70Met and Arg70Gln mutant enzymes. It is proposed that the electrostatic interaction between the guanidinium group of Arg 70 and the carboxyl group of PEP is important for PEP binding and for further steps in catalysis.
Limited proteolysis of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase
Journal of Protein Chemistry, 1993
Incubation ofSaccharomyces cerevisiae phosphoenolpyruvate carboxykinase with trypsin under native conditions cases a time-dependent loss of activity and the production of protein fragments. Cleavage sites determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and sequence analyses identified protease-sensitive peptide bonds between amino acid residues at positions 9–10 and 76–77. Additional fragmentation sites were also detected in a region approximately 70–80 amino acids before the carboxyl end of the protein. These results suggest that the enzyme is formed by a central compact domain comprising more than two thirds of the whole protein structure. From proteolysis experiments carried out in the presence of substrates, it could be inferred that CO2 binding specifically protects position 76–77 from trypsin action. Intrinsic fluorescence measurements demonstrated that CO2 binding induces a protein conformational change, and a dissociation constant for the enzyme CO2 complex of 8.2±0.6 mM was determined
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
The Protein Journal, 2007
The kinetic affinity for CO 2 of phosphoenolpyruvate PEP 5 carboxykinase from Anaerobiospirillum succiniciproducens, an obligate anaerobe which PEP carboxykinase catalyzes the carboxylation of PEP in one of the final steps of succinate production from glucose, is compared with that of the PEP carboxykinase from Saccharomyces cerevisiae, which catalyzes the decarboxylation of oxaloacetate in one of the first steps in the biosynthesis of glucose. For the A. succiniciproducens enzyme, at physiological concentrations of Mn 2+ and Mg 2+ , the affinity for CO 2 increases as the ATP/ADP ratio is increased in the assay medium, while the opposite effect is seen for the S. cerevisiae enzyme. The results show that a high ATP/ADP ratio favors CO 2 fixation by the PEP carboxykinase from A. succiniciproducens but not for the S. cerevisiae enzyme. These findings are in agreement with the proposed physiological roles of S. cerevisiae and A. succiniciproducens PEP carboxykinases, and expand recent observations performed with the enzyme isolated from Panicum maximum (Chen et al. (2002) Plant Physiology 128: 160-164).
Functional evaluation of serine 252 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase
Biochimie, 2009
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase mutant Ser252Ala, affecting the conserved Walker A serine residue, was characterized to elucidate the role of this serine residue. The substitution did not result in changes in the protein structure, as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analysis of the mutated enzyme in both directions of the main reaction and in the two secondary reactions showed an approximately 50-fold increase in apparent K m for oxaloacetate with minor alterations in the other kinetic parameters. These results show that the hydroxyl group of serine 252 is required for proper oxaloacetate interaction.
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.
Archives of Biochemistry and Biophysics, 1988
Saccharcwnyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.491 is completely inactivated by the 2',3'dialdehyde derivative of ATP (oATP) in the presence of Mn'+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 f 17 PM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30°C is 0.200 + 0.045 min-'. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered K, but a decreased Vas compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pK, of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase. o 198s Academic PM, I~C.