The pyruvate kinase (PYK) is the enzyme that catalyzes the last step in the glycolytic pathway, which involves the irreversible transfer of the phosphate group of one molecule of phosphoenolpyruvate (PEP) to a molecule of ADP, resulting in the synthesis of a molecule of ATP and another of pyruvic acid or pyruvate.
The pyruvate thus produced subsequently participates in various catabolic and anabolic (biosynthetic) pathways: it can be decarboxylated to produce acetyl-CoA, carboxylated to produce oxaloacetate, transaminate to produce alanine, oxidized to produce lactic acid or it can be directed towards gluconeogenesis for synthesis glucose.
Reaction catalyzed by the enzyme pyruvate kinase (Source: Noah Salzman via Wikimedia Commons)
Since it participates in glycolysis, this enzyme is very important for the carbohydrate metabolism of many organisms, unicellular and multicellular, which use this as the main catabolic route for obtaining energy.
An example of cells strictly dependent on glycolysis for energy production is that of mammalian erythrocytes, for which a deficiency in any of the enzymes involved in this pathway can have considerably negative effects.
Structure
Four isoforms of the pyruvate kinase enzyme have been described in mammals:
- PKM1, typical in muscles
- PKM2, only in fetuses (both products of alternative processing of the same messenger RNA)
- PKL, present in the liver and
- PKR, present in erythrocytes (both encoded by the same gene, PKLR, but transcribed by different promoters).
However, the analyzes carried out on the structure of the different pyruvate kinase enzymes in nature (including these 4 from mammals) show a great similarity in the general structure, as well as with respect to the architecture of the active site and the regulatory mechanisms.
In general terms, it is an enzyme with a molecular weight of 200 kDa, characterized by a tetrameric structure composed of 4 identical protein units, of more or less 50 or 60 kDa, and each one with 4 domains, namely:
- A small helical domain at the N-terminal end (absent in bacterial enzymes)
- An “ A ” domain, identified by a topology of 8 folded β sheets and 8 α helices
- A " B " domain, inserted between folded beta sheet number 3 and alpha helix number 3 of domain "A"
- A " C " domain, which has an α + β topology
Molecular structure of the pyruvate kinase enzyme (Source: Jawahar Swaminathan and MSD staff at the European Bioinformatics Institute via Wikimedia Commons)
Three sites have been detected in pyruvate kinase tetramers from different organisms: the active site, the effector site, and the amino acid binding site. The active site of these enzymes is located between domains A and B, in the vicinity of the "effector site", which belongs to domain C.
In the tetramer, the C domains form a "small" interface, while the A domains form a larger interface.
Function
As already discussed, pyruvate kinase catalyzes the last step in the glycolytic pathway, that is, the transfer of a phosphate group from phosphoenolpyruvate (PEP) to an ADP molecule to produce ATP and a pyruvate or pyruvic acid molecule.
The products of the reaction catalyzed by this enzyme are of utmost importance for different metabolic contexts. Pyruvate can be used in different ways:
- Under aerobic conditions, that is, in the presence of oxygen, this can be used as a substrate for an enzyme known as the pyruvate dehydrogenase complex, to be decarboxylated and converted into acetyl-CoA, a molecule that can enter the Krebs cycle in the mitochondria or participate in other anabolic pathways such as fatty acid biosynthesis, for example.
- In the absence of oxygen or anaerobiosis, pyruvate can be used by the enzyme lactate dehydrogenase to produce lactic acid (oxidation) through a process known as "lactic fermentation".
- In addition, pyruvate can be converted into glucose through gluconeogenesis, into alanine through alanine transaminase, into oxaloacetate through pyruvate carboxylase, etc.
It is important to remember that in the reaction catalyzed by this enzyme, the net synthesis of ATP also occurs, which is accounted for for glycolysis, producing 2 molecules of pyruvate and 2 molecules of ATP for each molecule of glucose.
Thus, from this perspective, the pyruvate kinase enzyme plays a fundamental role in many aspects of cellular metabolism, so much so that it is used as a therapeutic target for many human pathogens, among which several protozoa stand out.
Regulation
Pyruvate kinase is an extremely important enzyme from the point of view of cellular metabolism, since it is the one that forms the last compound resulting from the glucose catabolism pathway: pyruvate.
In addition to being one of the three most regulated enzymes in the entire glycolytic pathway (the other two being hexokinase (HK) and phosphofructokinase (PFK)), pyruvate kinase is a very important enzyme for the control of metabolic flow and production of ATP through glycolysis.
It is activated by phosphoenolpyruvate, one of its substrates (homotropic regulation), as well as by other mono- and diphosphorylated sugars, although its regulation depends on the type of isoenzyme considered.
Some scientific texts suggest that the regulation of this enzyme also depends on its "multidomain" architecture, since its activation seems to depend on some rotations in the domains of the subunits and on alterations in the geometry of the active site.
For many organisms, allosteric activation of pyruvate kinase is dependent on fructose 1,6-bisphosphate (F16BP), but this is not true for plant enzymes. Other enzymes are also activated by cyclic AMP and glucose 6-phosphate.
Furthermore, it has been shown that the activity of most of the pyruvate kinases studied is highly dependent on the presence of monovalent ions such as potassium (K +) and divalent ions such as magnesium (Mg + 2) and manganese (Mn + 2).).
Inhibition
Pyruvate kinase is mainly inhibited by physiological allosteric effectors, so these processes vary considerably between different species and even between types of cells and tissues of the same organism.
In many mammals, glucagon, epinephrine, and cAMP have inhibitory effects on pyruvate kinase activity, effects that can be counteracted by insulin.
Furthermore, it has been proven that some amino acids, such as phenylalanine, can act as competitive inhibitors for this enzyme in the brain.
References
- Morgan, HP, Zhong, W., McNae, IW, Michels, PA, Fothergill-Gilmore, LA, & Walkinshaw, MD (2014). Structures of pyruvate kinases display evolutionarily divergent allosteric strategies. Royal Society open science, 1 (1), 140120.
- Schormann, N., Hayden, KL, Lee, P., Banerjee, S., & Chattopadhyay, D. (2019). An Overview of Structure, Function and Regulation of Pyruvate Kinases. Protein Science.
- Valentini, G., Chiarelli, L., Fortin, R., Speranza, ML, Galizzi, A., & Mattevi, A. (2000). The allosteric regulation of pyruvate kinase A site-directed mutagenesis study. Journal of Biological Chemistry, 275 (24), 18145-18152.
- Valentini, G., Chiarelli, LR, Fortin, R., Dolzan, M., Galizzi, A., Abraham, DJ,… & Mattevi, A. (2002). Structure and function of human erythrocyte pyruvate kinase Molecular basis of nonspherocytic hemolytic anemia. Journal of Biological Chemistry, 277 (26), 23807-23814.
- Israelsen, WJ, & Vander Heiden, MG (2015, July). Pyruvate kinase: function, regulation and role in cancer. In Seminars in cell & developmental biology (Vol. 43, pp. 43-51). Academic Press.