Hydrolysis of phosphoenolpyruvate catalyzed by phosphoenolpyruvate carboxylase from Zea mays.

In addition to the normal carboxylation reaction, phosphoenolpyruvate carboxylase from Zea mays catalyzes a HCO3(-)-dependent hydrolysis of phosphoenolpyruvate to pyruvate and Pi. Two independent methods were used to establish this reaction. First, the formation of pyruvate was coupled to lactate dehydrogenase in assay solutions containing high concentrations of L-glutamate and aspartate aminotransferase. Under these conditions, oxalacetic acid produced in the carboxylation reaction was efficiently transaminated, and decarboxylation to form spurious pyruvate was negligible. Second, sequential reduction of oxalacetate and pyruvate was achieved by initially running the reaction in the presence of malate dehydrogenase with NADH in excess over phosphoenolpyruvate. After the reaction was complete, lactate dehydrogenase was added, thus giving a measure of pyruvate concentration. At pH 8.0 in the presence of Mg2+, the rate of phosphoenolpyruvate hydrolysis was 3-7% of the total reaction rate. The hydrolysis reaction catalyzed by phosphoenolpyruvate carboxylase was strongly metal dependent, with rates decreasing in the order Ni2+ greater than Co2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. These results suggest that the active site metal ion binds to the enolate oxygen, thus stabilizing the proposed enolate intermediate. The more stable the enolate, the less reactive it is toward carboxylation and the greater the opportunity for hydrolysis.     

A simplified assay for phosphoenolpyruvate.

A new method for analysis of phosphoenolpyruvate has been developed. The assay is based upon the stoichiometric conversion of ADP to ATP by the enzyme pyruvate kinase in the presence of variable amounts of PEP, and subsequent measurement of the ATP with a luciferin-luciferase preparation.     

Inactivation of phosphoenolpyruvate carboxykinase by acetaldehyde.

Preincubation with acetaldehyde at 37°C inactivates rat liver phosphoenolpyruvate carboxykinase. The inactivation is dependent upon the acetaldehyde concentration and the pH and duration of preincubation, and is prevented but not reversed by glutathione. The binding of the substrate ITP appears to be affected in the inactivation process. This effect of acetaldehyde might contribute to inhibition of gluconeogenesis resulting from ethanol metabolism.     

Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

The stereochemistry of the carboxylation of phosphoenolpyruvate to yield oxalacetate, catalyzed by chicken liver phosphoenolpyruvate carboxykinase and by Ascaris muscle phosphoenolpyruvate carboxykinase, was determined. The substrate (Z)-3-fluorophosphoenolpyruvate was used for the stereochemical analysis. The carboxylation reaction was coupled to malate dehydrogenase to yield 3-fluoromalate, and the stereochemistry of the products was identified by 19F NMR. In separate experiments, the enantiomeric tautomers of 3-fluorooxalacetate were shown to be utilized by malate dehydrogenase to yield (2R,3R)- and (2R,3S)-3-fluoromalate in nearly identical amounts. The products were identified by 19F NMR. When (Z)-3-fluorophosphoenolpyruvate was used as a substrate for phosphoenolpyruvate carboxykinase from avian liver and from Ascaris, and malate dehydrogenase was used to trap the product, only a single diastereomer was observed. This product was shown to be (2R,3R)-3-fluoromalate in each case. The assignments were based on coupling constants taken from Keck et al. [Keck, R., Hess, H., & Rétey, J. (1980) FEBS Lett. 114, 287]. These results indicate that the stereochemistry of carboxylation, catalyzed by chicken phosphoenolpyruvate carboxykinase and by Ascaris phosphoenolpyruvate carboxykinase, is identical and takes place from the si side of the enzyme-bound phosphoenolpyruvate. The carboxylation reaction was run both in H2O and in D2O. No deuterium incorporation into fluoromalate was shown to occur. The product 3-fluorooxalacetate is thus released from phosphoenolpyruvate carboxykinase as the keto form and is reduced more rapidly by reduced nicotinamide adenine dinucleotide with malate dehydrogenase than by the occurrence of tautomerization.     

Alteration of growth yield by overexpression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in Escherichia coli.

Phosphoenolpyruvate and oxaloacetate are key intermediates at the junction between catabolism and biosynthesis. Alteration of carbon flow at these branch points will affect the growth yield and the formation of products. We attempted to modulate the metabolic flow between phosphoenolpyruvate and oxaloacetate by overexpressing phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase from a multicopy plasmid under the control of the tac promoter. It was found that overexpression of phosphoenolpyruvate carboxylase decreased the rates of glucose consumption and organic acid excretion, but the growth and respiration rates remained unchanged. Consequently, the growth yield on glucose was improved. This result indicates that the wild-type level of phosphoenolpyruvate carboxylase is not optimal for the most efficient glucose utilization in batch cultures. On the other hand, overexpression of phosphoenolpyruvate carboxykinase increased glucose consumption and decreased oxygen consumption relative to those levels required for growth. Therefore, the growth yield on glucose was reduced because of a higher rate of fermentation product excretion. These data provide useful insights into the regulation of central metabolism and facilitate further manipulation of pathways for metabolite production.     

A microtiter plate-based assay for phosphoenolpyruvate carboxylase.

A sensitive, quantitative assay for phosphenolpyruvate carboxylase which utilizes microtiter plates is described. The assay depends upon the production of a colored compound in the reaction between oxaloacetate, the product of the phosphoenolpyruvate reaction, and the dye Fast Violet B. The method is particularly appropriate for monitoring chromatographic eluates and its utility for this purpose is demonstrated by the detection of phosphoenolpyruvate carboxylase in fractions of crude maize extract separated by size-exclusion chromatography.     

Transport of phosphoenolpyruvate through the erythrocyte membrane.

Phosphoenolpyruvate was transported through the erythrocyte membrane at low pH (4.5-6.5). The influx was observed not only in an iso-osmotic sucrose medium, but also in 0.1 M-citrate solution, but it was negligible in an iso-osmotic NaC1 solution. Efflux, however, was observed in both the sucrose and NaC1 solutions. Compounds derived from phosphoenolpyruvate by replacing the methene group by similarly hydrophobic groups such as hydrogen or the methyl group were permeant but those with the hydrophilic hydroxymethyl group were impermeant. This transport was inhibited by the treatment with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid or pyridoxal phosphate/NaBH4, which are known to be specific for the transport of anions such as C1-, SO42- and HPO42-. It showed saturation kinetics with respect to phosphoenolpyruvate concentration in the medium. These results suggest that the transport of phosphoenolpyruvate is mediated by the anion-transport system. Although phosphoenolpyruvate was transported against the concentration gradient, the transport was characterized as a passive transport, and this apparent uphill transport was interpreted by the Donnan equilibrium.     

Assay of phosphoenolpyruvate carboxykinase in crude yeast extracts.

The applicability of a spectrophotometric assay of phosphoenolpyruvate car?ykinase to crude yeast extracts has been studied. The assay measured oxalacetate production by coupling to the malate dehydrogenase reaction (phosphoenolpyruvate + ADP + bicarbonate → oxalacetate + ATP; oxalacetate + NADH → malate + NAD). Disappearance of NADH depended strictly on the presence of phosphoenolpyruvate, bicarbonate, ADP, and Mn²⁺. Furthermore, the disappearance of NADH was shown to be accompanied by stoichiometric accumulation of malate. Addition of 10 mm quinolinate, which is a known inhibitor of liver phosphoenolpyruvate car?ykinase, completely prevented phosphoenolpyruvate-dependent NADH disappearance. These observations demonstrated that the assay provides a quantitative measure of phosphoenolpyruvate car?ykinase activity in crude extracts. The assay could be applied to crude extracts from yeast cells grown under laboratory conditions but not to extracts from commercially produced baker's yeast, because of an extremely high rate of endogeneous oxidation of NADH in the latter extracts. With the spectrophotometric assay, optimal activity was observed at pH 7.0 with both crude extracts and a 15-fold-purified preparation.     

Carboxylation of phosphoenolpyruvate by extracts of Neisseria gonorrhoeae.

The enzymatic carboxylation of phosphoenolpyruvate by cell-free extracts of Neisseria gonorrhoeae was examined and determined to be similar to the reaction catalyzed by phosphoenolpyruvate carboxylase (PEPC). This was shown by the irreversibility of the reaction and nucleotide independency. The enzyme was found to have some characteristics different from the other bacterial PEPCs reported. The enzyme showed catalytic activity in the presence of cobalt ions as well as magnesium and manganese ions, was not inhibited by succinate in fresh extracts, and displayed a low Michaelis constant for bicarbonate (0.27 mM), as compared with other PEPCs. The significance of this low Michaelis constant is discussed with respect to the growth of the organism and the importance of this enzyme to protein and nucleic acid synthesis.     

Regulation at the phosphoenolpyruvate branchpoint in Azotobacter vinelandii: phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Azotobacter vinelandii, like the corresponding enzyme from other organisms, is activated by acetyl coenzyme A and inhibited by l-aspartate. Both modifiers affect primarily the affinity of the enzyme for phosphoenolpyruvate. This is the first enzyme with a strictly anaplerotic (intermediate-replacing) function to be tested for response to the adenylate energy charge; it is entirely insensitive to variation in charge. The results suggest that carboxylation of phosphoenolpyruvate in this organism is controlled by negative feedback from aspartate and by the stimulatory effect of acetyl coenzyme A. The adenylate energy charge may be expected to affect the rate of this reaction indirectly through its effects on the concentrations of acetyl coenzyme A and l-aspartate.     

Plausible phosphoenolpyruvate binding site revealed by 2.6 A structure of Mn2+-bound phosphoenolpyruvate carboxylase from Escherichia coli.

We have determined the crystal structure of Mn2+-bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X-ray diffraction at 2.6 A resolution, and specified the location of enzyme-bound Mn2+, which is essential for catalytic activity. The electron density map reveals that Mn2+ is bound to the side chain oxygens of Glu-506 and Asp-543, and located at the top of the alpha/beta barrel in PEPC. The coordination sphere of Mn2+ observed in E. coli PEPC is similar to that of Mn2+ found in the pyruvate kinase structure. The model study of Mn2+-bound PEPC complexed with phosphoenolpyruvate (PEP) reveals that the side chains of Arg-396, Arg-581 and Arg-713 could interact with PEP.     

Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase. Mutagenesis at metal site 1

Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate (OAA) and ATP from PEP, ADP and CO(2). Mutations of PEP carboxykinase have been constructed where the residues His(225) and Asp(263), two residues of the enzyme's putative Mn(2+) binding site, were altered. Kinetic studies of the His225Glu, and Asp263Glu PEP carboxykinases show 600- and 16,800-fold reductions in V(max) relative to the wild-type enzyme, respectively, with minor alterations in K(m) for Mn(2+). Molecular modeling of wild-type and mutant enzymes suggests that the lower catalytic efficiency of the Asp263Glu enzyme could be explained by a movement of the lateral chain of Lys(248), a critical catalytic residue, away from the reaction center. The effect on catalysis of introducing a negatively charged oxygen atom in place of N(epsilon-2) at position 225 is discussed in terms of altered binding energy of the intermediate enolpyruvate.