Pyrophosphatases

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Pyrophosphatases
Pyrophosphatases
Fig. 1: Left : Inorganic pyrophosphatase (PPi).
Right : Diphosphatase ("organic pyrophosphatase", oP), R 1 = organic residue, R 2 = organic residue or H.
Enzyme classification
EC, category 3.6.1.- ,  hydrolases
Response type Exergonic hydrolysis
Substrate Anions or organic derivatives of diphosphoric acid
Products Anions or organic derivatives of phosphoric acid
Occurrence
Parent taxon Creature

As pyrophosphatase are mostly enzymes called the anions of diphosphoric ( pyrophosphate ) to phosphate split. The currently valid name for these pyrophosphatases is inorganic diphosphatases . PPase is the common abbreviation that will be used in the following.

In addition, (organic) diphosphatases, which split (organic) derivatives of pyrophosphoric acid with absorption of water , are also referred to as pyrophosphatases.

Inorganic Diphosphatases (PPases)

Fig. 2: The membrane-bound ATP synthase coupled to a cation flow (+) produces ATP from ADP and inorganic phosphate (Pi). In many anabolic reactions, these two starting materials are returned (black arrows). In contrast, a number of other processes produce pyrophosphate (PPi) and AMP . The latter is converted to ADP by an adenylate kinase with consumption of ATP. Different placed diphosphatases (cPPase, vPPase and mPPase) can be used to regenerate phosphate.

Inorganic diphosphate (pyrophosphate, PPi) is formed in all biosyntheses of macromolecules (proteins, DNA, RNA, cell walls, cellulose, etc.).

Its cleavage to phosphate is necessary in order to regenerate this substrate of the ATP synthase . In addition, the diphosphate concentration in the cytosol must be kept low so that the reactions that cause it do not stall.

The hydrolysis of pyrophosphate (PPi) to phosphate (Pi) catalyzed by PPases is exergonic :

PPi + H 2 O ⇌ 2 Pi
ΔG 0 ' = −20-25 kJ / mol

Pyrophosphatases are found in all living things, from bacterial cells to human organisms. There are three different groups of PPases, which differ fundamentally in their structure, their evolution and their reaction mechanism.

Compared to the slow, uncatalyzed hydrolysis, all pyrophosphatases increase the rate of diphosphate cleavage by many orders of magnitude. However, the mPPases and vPPases integrated in the membrane are significantly slower.

They all need Mg 2+ ions for their function . In the reaction center, these form the links between negatively charged amino acid groups of the enzyme and the two substrates pyrophosphate and water. Because the reaction centers in the three groups are structured very differently, they react differently to selective inhibitors . They are competitively inhibited by the pyrophosphate analog AMDP (aminomethylene diphosphonate, PO 3 -CHNH 2 -PO 3 4- ) at different concentrations , as well as by fluoride , which can interfere with the H 2 O binding site.

cPPases I cPPases II mPPases and vPPases
Reaction rate k cat (s −1 ) 200-400 1700-3000 3.5-20
k i AMDP inhibition (µM) 11-150 1000 1.2-1.8
k i fluoride - inhibition (uM) 11-90 6th 3000-4800

Group I cPPases

Fig. 3: Distribution and evolution of cPPases group I (status 1999), further explanations in the text.

Soluble cPPases of group I are found in all three domains of living things. These include the cPPases from Escherichia coli (EPPase) and the baker's yeast Saccharomyces cerevisiae (YPPase). Both have long been known and have been intensively studied. The eukaryotic cPPases are derived from prokaryotic ancestors and are larger. The cPPase from Escherichia coli forms a hexamer ( i.e. six protein domains). eukaryotic cPPases, e.g. B. those from human cells and yeast cells form dimers .

In Fig. 3, bacteria are shown in red and archaea in purple. In the meantime it has been shown that the cPPases of archaea and bacteria form two separate groups. The vegetable (green) cPPases, which are closely related to each other, are clearly separated from this. The animals belonging to the Opisthokonta (blue) and the fungus S. cervisiae (cyan) have closely related cPPases.

It is noteworthy that mitochondria have their own group I cPPases. This also applies to the mitochondrial PPase of S. cervisiae (shown in red). The figure does not show the mitochondrial and cytosolic cPPase of the mouse, both of which can also be classified in the opisthokonta group. In many plants, group I cPPases are not found in the cytosol, but in the mitochondria and plastids . The diphosphate concentration in the cytosol can assume values ​​of 0.2–0.3 mM and is used there energetically by vPPases (see below).

Inorganic cPPase I
Inorganic cPPase I
Fig. 4. Ribbon model of cPPase I ( Thermococcus litoralis)
Identifier
External IDs
Enzyme classification
EC, category 3.6.1.1

The Thermus thermophilus bacterium has a temperature-resistant pyrophosphatase suitable for the polymerase chain reaction (PCR). During the PCR, the polymerization of the deoxyribonucleoside triphosphate (dNTP) produces pyrophosphate, which can have an inhibiting effect on the process. By breaking down pyrophosphate, the efficiency and yield of the PCR can be increased.

Group II cPPases

Group II cPPases are only found in prokaryotes , especially in bacterial firmicutes (Bacilli and Clostridia). The few representatives of these cPPases outside of this group have probably reached the organisms through horizontal gene transfer . They need Manganese 2+ (or Co 2+ ) to function. These enzymes hydrolyze pyrophosphate in the nanomolar range. Mn 2+ presumably causes their higher affinity for their substrates and their higher turnover rate. It is assumed that it increases the nucleophilicity of the hydrolyzing H 2 O and is also responsible for the greater sensitivity to F - .

The cPPase of the pathogenic bacterium Staphylococcus aureus belongs to group II. It is intensively investigated as a possible target for newly developed antibiotics.

Membrane-bound energy conserving mPPases and vPPases

Chemiosmotic function

The membrane-bound pyrophosphatases (mPPases and vPPases) differ not only structurally but also functionally from the cPPases which hydrolyze pyrophosphate dissolved in the cytosol. They couple the hydrolysis of PPi to the export of H + or Na + cations from the cytosol through a membrane:

PPi + cation + inside + H 2 O ⇌ 2 Pi + cation + outside

The exergonic hydrolysis is linked to an endergonic process, namely the transport of ions against a membrane potential . The potential energy contained therein increases due to the coupled hydrolysis. Part of the energy contained in the pyrophosphate is therefore conserved in the membrane potential.

The membrane-bound PPases not only serve as ion pumps , because the process is reversible. When the cations flow back, pyrophosphate is produced. After substrate chain phosphorylation and ATP formation by the ATP synthases, this reverse reaction is a third way of building high-energy phosphate bonds.

Occurrence

The chemiosmotic coupling of pyrophosphate synthesis to a proton gradient was proven back in 1966, when membrane vesicles of a phototrophic bacterium of the Rhodospirillaceae family converted phosphate to pyrophosphate. At that time, a synthesis of an energy-rich phosphate compound made possible by a membrane potential was only known from the ATP synthase , which, however, has a completely different structure than the PPase. However, there is apparently still a functional difference in Rhodospirillum to ATP synthase, because its PPase is primarily not located in the cell membrane, like that in the cell membrane, but in the membrane of the acidocalcisomes (acid organelles containing polyphosphate ).

Membrane PPases are found in 25% of all prokaryotes . In contrast, they do not seem to occur in fungi and multicellular animals. They play an essential role as vPPases in animal protozoa such as the pathogen Trypanosoma brucei , unicellular algae and especially in all land plants . They are particularly common in organisms that regularly suffer from lack of energy, oxygen, salt and other stressful conditions.

evolution

Fig. 5. MPPase of the bacterium Thermotoga maritima (top) and the mung bean Vigna radiata (bottom). P = periplasm, M = membrane, C = cytosol, V = vacuole

The mPPases and vPPases basically have the same structure (see Fig. 5) and a common evolutionary root that goes back to the ancestor of all living beings. It is also believed that they originate from a phase of chemical evolution when abiotic pyrophosphate perhaps played the central role as an energy carrier for the formation of nucleic acids .

The first membrane-bound PPase probably transported Na + . This is suggested by the comparative study of the amino acid sequences of these enzymes. The same assumption, namely that Na + pumps preceded the H + pumps, was also made for the ATP synthases . Biomembrane are generally easier for H + ions to penetrate than for Na + , and it required a whole time of the co-evolution of membrane proteins and membranes, until a H + -ATP synthase could function. From the Na + PPases, various H + -PPases have developed in their further evolution , some of which require K + for their functioning.

Molecular structure and reaction mechanism

Since 2012, the exact structure of a Na + -mPPase of the bacterium Thermotoga maritima and a plant-based H + -dependent vPPase that is evolutionarily distant from it could be determined. There is essentially no difference between the two (Fig. 5). They both consist of two functionally identical protein chains. These penetrate the membrane in 16 segments. Their reactive center is on the cytosolic side about 20 Å away from the membrane in a hydrophilic funnel-shaped structure.

In Fig. 5 the bacterial mPPase and the plant vPPase are shown schematically. In the front view, the dimeric enzymes are 85 Å wide and 75 Å high.

The reactive center is on the side of the cytosol below the membrane. Both enzymes offer hydrophilic access to the enzyme on the cytosol side and a hydrophilic channel at the top (arrow) through which the cations can penetrate the membrane.

The substrates are shown in the reactive center. Pyrophosphate is coupled to both proteins with 4 Mg + ions. The water used for hydrolysis is associated with the enzyme through two aspartic acid groups (not shown in Fig. 5).

Fig. 6. Possible reaction mechanism

Fig. 6 shows a working model for the reversible, ion-transporting hydrolysis. The hydrolytic path (clockwise in Fig. 6) can be imagined as follows:

  1. The pump mechanism is prepared by a pyrophosphate ion penetrating the reaction center in exchange for a hydrogen phosphate ion. In the PP phase, the enzyme is closed on the cytosolic side, but the transmembrane channel is briefly open.
  2. The hydrolysis takes place synchronously with the escape of the cation to the outside. An H + initially bound to aspartic acid D287 moves to location D294 to which the cation was previously bound. The OH - group remaining on D273 is so nucleophilic that the nucleophilic substitution to two hydrogen phosphate ions is possible ( 2P in the figure).
  3. In stage P , the enzyme after replacement of the first phosphate ion is a hydroxyl ion, OH - open bottom. A cation to be transported can penetrate. Then the state is restored in which the second phosphate ion can be replaced by pyrophosphate and a new cycle begins.

Each of these steps is reversible. If there is hardly any pyrophosphate present, the probability increases in the P + phase that the second phosphate will not be exchanged. After the cation has escaped inwards, a second phosphate (in exchange for OH - ) can penetrate the enzyme. A high concentration of cations on the outside then increases the chance that the condensation reaction to pyrophosphate will succeed.

The slow reaction rate in comparison to the cPPases results from the reversibility of the partial steps. The weak fluoride inhibition mentioned above (only 1 millimolar range) is probably due to the weak binding of the substrate H 2 O.

Organic diphosphatases

Fig. 7. Organic pyrophosphatases

"Pyrophosphatases" are sometimes also called hydrolases, which split organic substrates . They usually follow the scheme above in the adjacent figure.

  • Nucleotide diphosphatases ( EC  3.6.1.9 ), alias dinucleotide pyrophosphatase, cleave dinucleotides such as FAD . This is hydrolyzed to FMN and AMP . In the figure, R 1 = flavin and R 2 = adenosine. These enzymes are mostly non-specific and hydrolytically disable a number of other coenzymes , such as B. NAD and CoA . UDP-glucose is also hydrolyzed .
  • The enzymes known as pyrophosphatases include the nucleoside diphosphatases ( EC  3.6.1.6 ). They also serve to break down coenzymes. These include the so-called thiamine pyrophosphatase . (R 1 = thiamine , R 2 = H.) An ADPase, alias adenosine pyrophosphatase (R 1 = adenosine ) breaks down ADP by splitting off phosphate to form AMP.
  • No longer IUBMB -compliant name dihydroneopterin - pyrophosphatase denotes a dihydroneopterin triphosphate diphosphatase EC  3.6.1.67 , does not cleave the inorganic pyrophosphate, but releases (figure below).

Web links

Individual evidence

  1. ↑ According to IUBMB , the designation (inorganic) diphosphatase applies to (inorganic) pyrophosphatases [1] [2]
  2. ↑ Jukka K. Heinonen: Biological role of inorganic pyrophosphate. Springer Science & Business Media (formerly Kluwer Academic Publ), Berlin 2001, p. 11 .
  3. ↑ a b Alexander A. Baykov, Anssi M. Malinen, Heidi H. Luoto, Reijo Lahti: Pyrophosphate-Fueled Na + and H + Transport in Prokaryotes . In: Microbiol. Mol. Biol. Rev. . 77, No. 2, 2013, pp. 267-276. doi : 10.1128 / MMBR.00003-13 .
  4. ↑ a b c d e f g h Tommi Kajandera, Juho Kellosalob & Adrian Goldman: Inorganic pyrophosphatases: One substrate, three mechanisms . In: FEBS Letters . 587, No. 13, 2013, pp. 1863–1869. doi : 10.1016 / j.febslet.2013.05.003 .
  5. ↑ a b Shih-Ming Lin, Jia-Yin Tsai, Chwan-Deng Hsiao, Yun-Tzu Huang, Chen-Liang Chiu, Mu-Hsuan Liu, Jung-Yu Tung, Tseng-Huang Liu, Rong-Long Pan & Yuh- Ju Sun: Crystal structure of a membrane-embedded H + -translocating pyrophosphatase . In: Nature . 484, No. 7394, 2012, pp. 399-403. doi : 10.1038 / nature10963 .
  6. ↑ a b c Heidi H. Luoto, Georgiy A. Belogurov, Alexander A. Baykov, Reijo Lahti, and Anssi M. Malinen: Na + -translocating Membrane Pyrophosphatases Are Widespread in the Microbial World and Evolutionarily Precede H + -translocating Pyrophosphatases . In: J. Biol. Chem. . 286, No. 11, 2011, pp. 21633-21642. doi : 10.1074 / jbc.M111.244483 .
  7. ↑ a b Toni Sivula, Anu Salminen, Alexey N. Parfenyev, Pekka Pohjanjoki, Adrian Goldman, Barry S. Cooperman, Alexander A. Baykov, Reijo Lahti: Evolutionary aspects of inorganic pyrophosphatase . In: FEBS Letters . 454, No. 1-2, 1999, pp. 75-80. doi : 10.1016 / S0014-5793 (99) 00779-6 .
  8. ↑ A Kunitz: CRYSTALLINE INORGANIC pyrophosphatase ISOLATED FROM BAKER'S YEAST. In: The Journal of General Physiology. 1952; 35 (3): 423-450.
  9. ↑ Lana J. McMillana, Nathaniel L. Hepowit & Julie A. Maupin-Furlow: Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents . In: Applied and Environmental Microbiology . 82, No. 2, 2016, pp. 538-548. doi : 10.1128 / AEM.03055-15 .
  10. ↑ María R Gómez ‐ García, Manuel Losada, Aurelio Serrano: Comparative biochemical and functional studies of family I soluble inorganic pyrophosphatases from photosynthetic bacteria . In: FEBS Journal . 274, No. 15, 2007, pp. 3948-3959. doi : 10.1111 / j.1742-4658.2007.05927.x .
  11. ↑ Marco Zancani, Valentino Casolo, Carlo Peresson, Giorgio Federici, Andrea Urbani, Francesco Macrı̀, Angelo Vianello: The β-subunit of pea stem mitochondrial ATP synthase exhibits PPiase activity . In: Mitochondrion . 3, No. 2, 2003, pp. 111-118. doi : 10.1016 / S1567-7249 (03) 00105-3 .
  12. ↑ Chathurada S. Gajadeera, Xinyi Zhang, Yinan Wei, Oleg V. Tsodikov: Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site . In: Journal of Structural Biology . 189, No. 2, 2015, pp. 81-86. doi : 10.1016 / j.jsb.2014.12.003 .
  13. ↑ Laura M. Barge, Ivria J. Doloboff, Michael J. Russell, David VanderVelde, Lauren M. White, Galen D. Stucky, Marc M. Baum, John Zeytounian, Richard Kidd, Isik Kanik: Pyrophosphate synthesis in iron mineral films and membranes simulating prebiotic submarine hydrothermal precipitates . In: Geochimica et Cosmochimica Acta . 128, 2014, p. 42705. doi : 10.1016 / j.gca.2013.12.006 .
  14. ↑ Herrick Baltscheffsky, Lars-Victor von Stedingk, Hans-Walter Heldt, Martin Klingenberg: Inorganic Pyrophosphate: Formation in Bacterial Photophosphorylation (Inorganic pyrophosphate is identified as the major product of photophosphorylation by isolated chromatophores from Rhodospirillum rubrum in the absence of added nucleotides.) . In: Science . 274, No. 3740, 1966, pp. 1120-1122.
  15. ↑ Jennifer Moyle, Roy Mitchell, Peter Mitchell: Proton-translocating pyrophosphatase of Rhodospirillum rubrum . In: FEBS Letters . 23, No. 2, 1972, pp. 233-236. doi : 10.1016 / 0014-5793 (72) 80349-1 .
  16. ↑ Roberto Docampo, Wanderley de Souza, Kildare Miranda, Peter Rohloff and Silvia Moreno NJ: Acidocalcisomes? conserved from bacteria to man . In: Nature Reviews Microbiology . 3, No. 3, 2005, pp. 251-261. doi : 10.1038 / nrmicro1097 .
  17. ↑ Manfredo Seufferheld, Christopher R. Lea, Mauricio Vieira, Eric Oldfield, and Roberto Docampo: The H + -pyrophosphatase of Rhodospirillum rubrum is predominantly located in Polyphosphate-rich Acidocalcisomes . In: J. Biol. Chem. . 279, 2004, pp. 51193-51202. doi : 10.1074 / jbc.M406099200 .
  18. ↑ a b c Juho Kellosalo, Tommi Kajander, Konstantin Kogan, Kisun Pokharel, Adrian Goldman: The Structure and Catalytic Cycle of a Sodium-Pumping Pyrophosphatase . In: Science . 337, No. 6093, 2012, pp. 473-476. doi : 10.1126 / science.1222505 .
  19. ↑ [3]
  20. ↑ Shih-Ming Lin, Jia-Yin Tsai, Chwan-Deng Hsiao, Yun-Tzu Huang, Chen-Liang Chiu, Mu-Hsuan Liu, Jung-Yu Tung, Tseng-Huang Liu, Rong-Long Pan & Yuh-Ju Sun : Crystal structure of a membrane-embedded H + -translocating pyrophosphatase . In: Nature . 484, No. 7394, 2012, pp. 399-403. doi : 10.1038 / nature10963 . Graphic cf. A working model for proton pumping in VrH + -PPase
  21. ↑ [4]
  22. ↑ Herrick Baltscheffsky: Energy conversion leading to the origin and early evolution of life: did inorganic pyrophosphate precede adenosine triphosphate? . In: Origin and evolution of biological energy conversion. . John Wiley & Sons, 1996.
  23. ↑ a b Tommi Kajandera, Juho Kellosalob & Adrian Goldman: Inorganic pyrophosphatases: One substrate, three mechanisms . In: FEBS Letters . 587, No. 13, 2013, p. 1866 f. doi : 10.1016 / j.febslet.2013.05.003 .
  24. ↑ arms Y. Mulkidjanian, Pavel Dibrov, Michael Y. Galperin: The past and present of sodium energetics: May the sodium-motive force be with you . In: Biochimica et Biophysica Acta (BBA) - Bioenergetics . 1777, No. 7-8, 2008, pp. 985-992. doi : 10.1016 / j.bbabio.2008.04.028 .
  25. ↑ Armen Y Mulkidjanian, Michael Y Galperin, Kira S Makarova, Yuri I Wolf and Eugene V Koonin: Evolutionary primacy of sodium bioenergetics . In: Biology Direct . 3, No. 13, 2008. doi : 10.1186 / 1745-6150-3-13 .
  26. ↑ arms Y Mulkidjanian, Kira Makarova S, Y Michael Galperin, Eugene V Koonin: Inventing the dynamo machine: the evolution of the F-type and V-type ATPases . In: Nature Reviews Microbiology . 5, No. 11, 2007, pp. 892-899. doi : 10.1038 / nrmicro1767 . This article at Uni Osnabrück: Perspectives (PDF)  ( page no longer available , search in web archives )  Info: The link was automatically marked as defective. Please check the link according to the instructions and then remove this notice.@1@ 2Template: Toter Link / www.macromol.uni-osnabrueck.de  
  27. ↑ Suzanne J. Admiraal & Daniel Herschlag: Mapping the transition state for ATP hydrolysis: implications for enzymatic catalysis . In: Chemistry & Biology . 2, No. 11, 1995, pp. 729-739. doi : 10.1016 / 1074-5521 (95) 90101-9 .
  28. ↑ Lucien Bettendorff & Pierre Wins: Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors . In: FEBS Journal . 276, No. 11, 2009, pp. 2917-2925. doi : 10.1111 / j.1742-4658.2009.07019.x .