Inorganic pyrophosphatase
Encyclopedia
Pyrophosphatase is an enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

 that catalyzes the conversion of one molecule of pyrophosphate
Pyrophosphate
In chemistry, the anion, the salts, and the esters of pyrophosphoric acid are called pyrophosphates. Any salt or ester containing two phosphate groups is called a diphosphate. As a food additive, diphosphates are known as E450.- Chemistry :...

 to two phosphate
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...

 ions. This is a highly exergonic
Exergonic
Exergonic means "releasing energy in the form of work". By thermodynamic standards, work, a form of energy, is defined as moving from the system to the surroundings...

 reaction, and therefore can be coupled to unfavorable biochemical transformations in order to drive these transformations to completion. The functionality of this enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

 plays a critical role in lipid metabolism
Lipid metabolism
Lipid metabolism refers to the processes that involve the intercourse and degradation of lipids.The types of lipids involved include:* Bile salts* Cholesterols* Eicosanoids* Glycolipids* Ketone bodies* Fatty acids - see also fatty acid metabolism...

 (including lipid synthesis and degradation), calcium absorption and bone formation, and DNA synthesis, as well as other biochemical transformations.

Structure

Thermostable pyrophosphatase has been isolated from the extremophile
Extremophile
An extremophile is an organism that thrives in physically or geochemically extreme conditions that are detrimental to most life on Earth. In contrast, organisms that live in more moderate environments may be termed mesophiles or neutrophiles...

 Thermococcus litoralis
Thermococcus litoralis
Thermococcus litoralis is a species of archaea.-Location:Thermococcus litoralis grows near and around deep-sea smoker vents.-Reproduction:...

. The 3-dimensional structure was determined using x-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...

, and was found to consist of two alpha-helices
Alpha helix
A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...

, as well as an antiparallel
Antiparallel (biochemistry)
In biochemistry, two molecules are antiparallel if they run side-by-side in opposite directions or when both strands are complimentary to each other....

 closed beta-sheet. The form of inorganic pyrophosphatase isolated from Thermococcus litoralis
Thermococcus litoralis
Thermococcus litoralis is a species of archaea.-Location:Thermococcus litoralis grows near and around deep-sea smoker vents.-Reproduction:...

was found to contain a total of 174 amino acid residues and have a hexameric oligomeric organization (Image 1). Though the human form of the enzyme has not yet been isolated, a 1.23 kilobase cDNA segment has been identified that encodes a 32 kDa protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 that is 94% identical to bovine inorganic pyrophosphatase. This DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 sequence has assigned to a gene locus on human chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...

 10.

Mechanism

Though the precise mechanism of catalysis
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....

 via inorganic pyrophosphatase in most organisms remains uncertain, site-directed mutagenesis
Mutagenesis
Mutagenesis is a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures...

 studies in Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

have allowed for analysis of the enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

 active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...

 and identification of key amino acids. In particular, this analysis has revealed 17 residues of that may be of functional importance in catalysis
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....

.

Further research suggests that the protonation
Protonation
In chemistry, protonation is the addition of a proton to an atom, molecule, or ion. Some classic examples include*the protonation of water by sulfuric acid:*the protonation of isobutene in the formation of a carbocation:2C=CH2 + HBF4 → 3C+ + BF4−*the protonation of ammonia in the...

 state of Asp67 is responsible for modulating the reversibility of the reaction
Chemical reaction
A chemical reaction is a process that leads to the transformation of one set of chemical substances to another. Chemical reactions can be either spontaneous, requiring no input of energy, or non-spontaneous, typically following the input of some type of energy, such as heat, light or electricity...

 in Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

. The hydroxide
Hydroxide
Hydroxide is a diatomic anion with chemical formula OH−. It consists of an oxygen and a hydrogen atom held together by a covalent bond, and carrying a negative electric charge. It is an important but usually minor constituent of water. It functions as a base, as a ligand, a nucleophile, and a...

 functional group of this residue has been shown to perform a nucleophilic attack on the pyrophosphate
Pyrophosphate
In chemistry, the anion, the salts, and the esters of pyrophosphoric acid are called pyrophosphates. Any salt or ester containing two phosphate groups is called a diphosphate. As a food additive, diphosphates are known as E450.- Chemistry :...

 substrate
Substrate (chemistry)
In chemistry, a substrate is the chemical species being observed, which reacts with a reagent. This term is highly context-dependent. In particular, in biochemistry, an enzyme substrate is the material upon which an enzyme acts....

 when four magnesium
Magnesium
Magnesium is a chemical element with the symbol Mg, atomic number 12, and common oxidation number +2. It is an alkaline earth metal and the eighth most abundant element in the Earth's crust and ninth in the known universe as a whole...

 ions are present. Direct coordination with these four magnesium
Magnesium
Magnesium is a chemical element with the symbol Mg, atomic number 12, and common oxidation number +2. It is an alkaline earth metal and the eighth most abundant element in the Earth's crust and ninth in the known universe as a whole...

 ions and hydrogen bonding interactions with Arg43, Lys29, and Lys142 (all positively-charged residues) have been shown to anchor the substrate to the active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...

. The four magnesium
Magnesium
Magnesium is a chemical element with the symbol Mg, atomic number 12, and common oxidation number +2. It is an alkaline earth metal and the eighth most abundant element in the Earth's crust and ninth in the known universe as a whole...

 ions are also suggested to be involved in the stabilization of the trigonal bipyramid transition state
Transition state
The transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...

, which lowers the energetic barrier for the aforementioned nucleophilic attack.

Several studies have also identified additional substrates that can act as allosteric effectors. In particular, the binding of pyrophosphate
Pyrophosphate
In chemistry, the anion, the salts, and the esters of pyrophosphoric acid are called pyrophosphates. Any salt or ester containing two phosphate groups is called a diphosphate. As a food additive, diphosphates are known as E450.- Chemistry :...

 (PPi) to the effector site of inorganic pyrophosphatase increases its rate of hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...

 at the active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...

. ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...

 has also been shown to function as an allosteric activator in Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

, while fluoride
Fluoride
Fluoride is the anion F−, the reduced form of fluorine when as an ion and when bonded to another element. Both organofluorine compounds and inorganic fluorine containing compounds are called fluorides. Fluoride, like other halides, is a monovalent ion . Its compounds often have properties that are...

 has been shown to inhibit hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...

 of pyrophosphate
Pyrophosphate
In chemistry, the anion, the salts, and the esters of pyrophosphoric acid are called pyrophosphates. Any salt or ester containing two phosphate groups is called a diphosphate. As a food additive, diphosphates are known as E450.- Chemistry :...

 in yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...

.

Biological Function and Significance

The hydrolysis of inorganic pyrophosphate
Pyrophosphate
In chemistry, the anion, the salts, and the esters of pyrophosphoric acid are called pyrophosphates. Any salt or ester containing two phosphate groups is called a diphosphate. As a food additive, diphosphates are known as E450.- Chemistry :...

 (PPi) to two phosphate
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...

 ions is utilized in many biochemical pathways to render reactions effectively irreversible. This process is highly exergonic
Exergonic
Exergonic means "releasing energy in the form of work". By thermodynamic standards, work, a form of energy, is defined as moving from the system to the surroundings...

 (accounting for approximately a −19kJ change in free energy
Thermodynamic free energy
The thermodynamic free energy is the amount of work that a thermodynamic system can perform. The concept is useful in the thermodynamics of chemical or thermal processes in engineering and science. The free energy is the internal energy of a system less the amount of energy that cannot be used to...

), and therefore greatly increases the energetic favorability of reaction system when coupled with a typically less-favorable reaction.

Inorganic pyrophosphatase catalyzes this hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...

 reaction in the early steps of lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...

 degradation, a prominent example of this phenomenon. By promoting the rapid hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...

 of pyrophosphate
Pyrophosphate
In chemistry, the anion, the salts, and the esters of pyrophosphoric acid are called pyrophosphates. Any salt or ester containing two phosphate groups is called a diphosphate. As a food additive, diphosphates are known as E450.- Chemistry :...

 (PPi), Inorganic pyrophosphatase provides the driving force for the activation of fatty acids destined for oxidation.

Before fatty acids can undergo degradation to fulfill the metabolic needs of an organism, they must first be activated via a thioester linkage to coenzyme A
Coenzyme A
Coenzyme A is a coenzyme, notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle. All sequenced genomes encode enzymes that use coenzyme A as a substrate, and around 4% of cellular enzymes use it as a substrate...

. This process is catalyzed by the enzyme acyl CoA synthetase, and occurs on the outer mitochondrial membrane. This activation is accomplished in two reactive steps: (1) the fatty acid reacts with a molecule of ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...

 to form an enzyme-bound acyl adenylate and pyrophosphate
Pyrophosphate
In chemistry, the anion, the salts, and the esters of pyrophosphoric acid are called pyrophosphates. Any salt or ester containing two phosphate groups is called a diphosphate. As a food additive, diphosphates are known as E450.- Chemistry :...

 (PPi), and (2) the sulfhydryl group of CoA attacks the acyl adenylate, forming acyl CoA and a molecule of AMP
Adenosine monophosphate
Adenosine monophosphate , also known as 5'-adenylic acid, is a nucleotide that is used as a monomer in RNA. It is an ester of phosphoric acid and the nucleoside adenosine. AMP consists of a phosphate group, the sugar ribose, and the nucleobase adenine...

. Each of these two steps is reversible under biological conditions, save for the additional hydrolysis of PPi by inorganic pyrophosphatase. This coupled hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...

 provides the driving force for the overall forward activation reaction, and serves as a source of inorganic phosphate used in other biological processes.

Evolution

Examination of prokaryotic and eukaryotic forms of inorganic pyrophosphatase has shown that they differ significantly in both amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

 sequence, number of residues, and oligomeric organization. Despite differing structural components, recent work has suggested a large degree of evolutionary conservation of active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...

 structure as well as reaction mechanism
Reaction mechanism
In chemistry, a reaction mechanism is the step by step sequence of elementary reactions by which overall chemical change occurs.Although only the net chemical change is directly observable for most chemical reactions, experiments can often be designed that suggest the possible sequence of steps in...

, based on kinetic
Kinetic energy
The kinetic energy of an object is the energy which it possesses due to its motion.It is defined as the work needed to accelerate a body of a given mass from rest to its stated velocity. Having gained this energy during its acceleration, the body maintains this kinetic energy unless its speed changes...

 data. Analysis of approximately one million genetic sequences taken from organisms in the Sargasso Sea
Sargasso Sea
The Sargasso Sea is a region in the middle of the North Atlantic Ocean, surrounded by ocean currents. It is bounded on the west by the Gulf Stream; on the north, by the North Atlantic Current; on the east, by the Canary Current; and on the south, by the North Atlantic Equatorial Current. This...

 identified a 57 residue sequence within the regions coding for inorganic pyrophosphatase that appears to be highly conserved; this region primarily consisted of the four early amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

 residues Gly, Ala
Ala
Ala may refer to:* Ala , a female demon in the Serbian and Bulgarian mythology* Ala , an Alusi in Odinani* Ala , a Republican-period, largely infantry, formation of 5,400 men...

, Val
Val
Val is a variant of the feminine given name Valerie.Val may refer to:-Given name:* Val Ackerman , American attorney, former basketball player, and first president of the Women's National Basketball Association* Val A...

 and Asp
Asp
Asp may refer to:Snakes:* Asp , an archaic term for a venomous snake.* Vipera aspis, a venomous viper found in Europe.* Cerastes cerastes, a venomous viper found in the Sahara desert....

, suggesting an evolutionarily ancient origin for the protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

.
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