Phosphoenolpyruvate carboxykinase


 * Not to be confused with Phosphoenolpyruvate carboxylase, PEPC

Phosphoenolpyruvate carboxykinase is an enzyme used in the natural process of gluconeogenesis.

Reversibility
While most reactions of gluconeogenesis simply use the glycolysis enzymes in the opposite direction, the pyruvate kinase enzyme is irreversible. Therefore, the enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase are used to provide an alternate path for effectively reversing its actions.

PEPCK in different species
PEPCK gene transcription (genetics) occurs in many species, and the amino acid sequence of PEPCK is distinct for each species.

For example, its structure and its specificity differ in humans, Escherichia coli (E. coli), and the parasite Trypanosoma cruzi.

In mammals, it is most abundant in the liver, kidney, and adipose tissue.

Researchers at Case Western Reserve University have discovered that overexpression of PEPCK in mice causes them to be more active, more aggressive, and have longer lives than normal mice; see metabolic supermice.

In an effort to explore the role of PEPCK, researchers caused the overexpression of PEPCK in E. coli bacteria via recombinant DNA.

Function in gluconeogenesis
It has been shown that PEPCK catalyzes the reversible rate-controlling step of gluconeogenesis, the process whereby glucose is synthesized. The enzyme has therefore been thought to be essential in glucose homeostasis, as evidenced by laboratory mice that contracted diabetes mellitus type 2 as a result of the overexpression of PEPCK.

A recent study suggests that the role that PEPCK plays in gluconeogenesis may be mediated by the citric acid cycle, the activity of which was found to be directly related to PEPCK abundance.

PEPCK levels alone were not found to be highly correlated with gluconeogenesis in the mouse liver, as previous studies have suggested. Therefore, the role of PEPCK in gluconeogenesis may be more complex and involve more factors than was previously believed.

PEPCK of Mycobacterium tuberculosis has been shown to trigger the immune system in mice by increasing cytokine activity.

As a result, it has been found that PEPCK may be an appropriate ingredient in the development of an effective subunit vaccination for tuberculosis.

PEPCK in plants and bacteria
PEPCK acts in plants that undergo C4 carbon fixation, where its action has been localized to the cytosol, in contrast to mammals, where it has been found that PEPCK works in mitochondria.

Although it is found in many different parts of plants, it has only been seen in specific cell types, including the areas of the phloem.

It has also been discovered that in cucumber (Cucumis sativus L.), PEPCK levels are increased by multiple effects that are known to decrease the cellular pH of plants, although these effects are specific to the part of the plant.

PEPCK levels rose in roots and stems when the plants were watered with ammonium chloride at a low pH (but not at high pH), or with butyric acid. However, PEPCK levels did not increase in leaves under these conditions.

In leaves, 5% CO2 content in the atmosphere lead to higher PEPCK abundance.

Structure
The structures formed when PEPCK complexes with other substances provide insight into the structure and also the mechanism of PEPCK enzymatic activity.

The three mitochondrial isoforms of PEPCK complex with Mn2+, Mn 2+ -phosphoenolpyruvate (PEP), and Mn2+-malonate- Mn2+ + GDP to give more information about its structure and how this enzyme catalyzes reactions.

Delbaere et al. (2004) resolved PEPCK in E. coli and found the active site sitting between a C-terminal domain and an N-terminal domain. The active site was observed to be closed upon rotation of these domains.

Phosphoryl groups are transferred during PEPCK action, which is likely facilitated by the eclipsed conformation of the phosphoryl groups when ATP is bound to PEPCK.

Since the eclipsed formation is one that is high in energy, phosphoryl group transfer has a decreased energy of activation, meaning that the groups will transfer more readily. This transfer likely happens via a mechanism similar to SN2 displacement.

Reaction Pathway
PEPCase converts oxaloacetate into phosphoenolpyruvate, and carbon dioxide.

As PEPCK acts at the junction between glycolysis and the Kreb’s cycle, it causes decarboxylation of a C4 molecule, creating a C3 molecule. As the first committed step in gluconeogenesis, PEPCK decarboxylates and phosphorylates oxaloacetate (OAA) for its conversion to PEP, when GTP is present. As a phosphate is transferred, the reaction results in a GDP molecule. Interestingly, when pyruvate kinase, the enzyme that normally catalyzes the reaction that converts PEP to pyruvate, is knocked out in mutants of Bacillus subtilis, PEPCK participates in one of the replacement anaplerotic reactions, working in the reverse direction of its normal function, converting PEP to OAA. Although this reaction is possible, the kinetics are so unfavorable that the mutants grow at a very slow pace or do not grow at all.

In fermentation, PEPCK catalyzes the reaction of PEP and carbon dioxide to OAA and ADP is therefore converted to ATP with the addition of a phosphate group.

In humans
PEPCK is enhanced, both in terms of its production and activation, by many factors. Transcription of the PEPCK gene is stimulated by glucagon, glucocorticoids, retinoic acid, and adenosine 3’,5’-monophosphate (cAMP), while it is inhibited by insulin. Of these factors, insulin, a hormone that is deficient in the case of diabetes, is considered dominant, as it inhibits the transcription of many of the stimulatory elements. PEPCK activity is also inhibited by hydrazine sulfate, and the inhibition therefore decreases the rate of gluconeogenesis.

In prolonged acidosis, PEPCK is upregulated in renal proximal tubule brush border cells, in order to secrete more NH3 and thus to produce more HCO3-.

The GTP-specific activity of PEPCK is highest when Mn2+ and Mg2+ are available. In addition, hyper-reactive cysteine (C307) is involved in the binding of Mn2+ to the active site.

Plants
As discussed previously, PEPCK abundance increased when plants were watered with low pH ammonium chloride, though high pH did not have this effect.

Classification
It is classified under EC number 4.1.1. There are three main types, distinguished by the source of the energy to drive the reaction:
 * 4.1.1.32 - GTP
 * 4.1.1.38 - diphosphate
 * 4.1.1.49 - ATP