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ALP

Official Full Name
ALP
Background
Alkaline phosphatase (ALP, ALKP, ALPase, Alk Phos) (EC 3.1.3.1) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment. It is sometimes used synonymously as basic phosphatase.
Synonyms
ALPP; Alkaline phosphatase Regan isozyme; Placental alkaline phosphatase 1; PLAP-1; Alkaline phosphatase; ALP; ALKP; ALPase; Alk Phos; EC 3.1.3.1; Alkaline phosphomonoesterase; Glycerophosphatase; Phosphomonoesterase

Catalog
ProductName
EC No.
CAS No.
Source
Price
CatalogNATE-1871
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceBovine Liver
CatalogNATE-1664
EC No.EC 3.1.3.1
CAS No.
SourceBaculovirus
CatalogNATE-1634
EC No.EC 3.1.3.1
CAS No.
SourceInsect cell (Ba...
CatalogEXWM-3607
EC No.EC 3.1.3.1
CAS No.9001-78-9
Source
CatalogNATE-0992
EC No.
CAS No.9001-78-9
SourcePichia pastoris
CatalogNATE-0947
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceHuman Liver
CatalogNATE-0946
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceBovine Kidney
CatalogNATE-0935
EC No.
CAS No.9001-78-9
Source
CatalogNATE-0807
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceProprietary hos...
CatalogNATE-0806
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceE. coli
CatalogNATE-0055
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceChicken Intesti...
CatalogNATE-0060
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceRabbit intestin...
CatalogNATE-0059
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourcePorcine kidney
CatalogNATE-0058
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourcePorcine intesti...
CatalogNATE-0057
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceHuman placenta
CatalogNATE-0056
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceEscherichia col...
CatalogNATE-0053
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceBovine intestin...
CatalogNATE-0061
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourcePichia pastoris
CatalogNATE-0054
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceCalf intestine
CatalogDIA-181
EC No.EC 3.1.3.1
CAS No.9001-78-9
SourceMicroorganism
Related Services
Related Protocols
PHOSPHATASE, ALKALINE -Enzymatic Assay Protocol
Related Reading

Alkaline phosphatase (ALP, EC 3.1.3.1) is a non-specific phospho-monoesterase that catalyzes the hydrolysis of virtually all phosphate monoesters to produce inorganic phosphates and the corresponding alcohols, phenols, and saccharides. It also catalyzes the transfer of phosphate groups, and E. coli ALP is also a phosphite-dependent hydrogenase. ALP exists in almost all living organisms except for higher plants and can directly participate in phosphorus metabolism, and plays an important role in the process of digestion, absorption, secretion, and ossification of calcium and phosphorus.

ALP

Molecular Structure

E. coli ALP is a homodimeric metalloprotease composed of two polypeptide chains, each containing 449 amino acids. There are three isoenzymes, isoenzyme I has an Arg residue at the N-terminus, isoenzyme III has no Arg residue at the N-terminus, and isoenzyme II is a heterodimer of these two forms. After being synthesized in cells, ALP monomers are localized in the periplasmic space and dimerized under the guidance of a leucine-rich N-terminal signal sequence, and become catalytically active enzyme molecules. E. coli ALP has one active site per monomer. The enzyme molecule exhibits an α/β topology, with 10 β-sheets in the center of the molecule and 15 α-helices of varying length on both sides. There is also a 3-layer β-sheet and a small α-helix on the top of the molecule. Each active site is a groove, opening in the enzyme surface. The active site has a strong affinity for phosphate, reaction products and competitive inhibitors, but there is no obvious binding site as the phosphoric acid monoester R group. The active site consists of Asp101-Ser102-Ala103, three spatially close metal ions and ligands, Arg166 and some other adjacent amino acid residues.

Catalytic Mechanism

ALP catalyzes the hydrolysis of phosphate monoesters or the transfer of phosphate groups. First, a covalent intermediate (E-P) is formed, and this covalent intermediate is subsequently hydrolyzed into a non-covalent complex (E•Pi). If a phosphor-receptor is present (such as Tris or ethanolamine), the enzyme will exhibit the activity of catalyzing the transfer of phosphate, transferring the phosphate group to the alcohol to form a new phosphate monoester. The rate-limiting step of the reaction is affected by the pH. Under acidic conditions, E-P hydrolysis is the rate-limiting step, and the rate-limiting step under alkaline conditions is the dissociation of the phosphate group in E.pi.

ALP Figure 1. ALP catalyzes the hydrolysis of phosphate monoesters or the transfer of phosphate groups. (Holtz K M; et al. 1999)

The active site of the enzyme is occupied by three water molecules, and the hydroxide ions coordinated with magnesium form hydrogen bonds with the hydroxyl oxygen of Ser102. Enzyme and substrate (ROP) combine to form a complex (E ▪ ROP). Ser102 is located opposite the dissociative group and requires deprotonation for nucleophilic attack of oxygen, for which reason it transfers the proton to the hydroxyl group of magnesium, forming the coordination of magnesium with water molecules. At the same time, Zn is also coordinated with the oxygen of Ser102, stabilizing it in a nucleophilic state. The catalytic mechanism of ALP involves two displacement reactions: Firstly, the activated Ser102 hydroxyl group attacks the phosphorus center of the E•ROP complex, forming a covalent enzyme phosphorous intermediate (E-Pi). The formation of E-Pi causes a change in the conformation of the phosphorous center and dissociation of the RO- group. Secondly, the nucleophilic hydroxide coordinated with Zn attacks E-Pi and hydrolyzes the phosphoserine intermediate to generate the non-covalent enzyme complex E·Pi. At the same time, the nucleophilic Ser102 regenerates and causes the second phosphorus center conformation change.

ALP Figure 2. Catalytic mechanism of ALP. (Holtz K M; et al. 1999)

Biological Functions

The exact biological functions of mammalian ALPs are not well understood. It is currently believed that bone mineralization is dependent on normal ALP activity. The most direct evidence for this viewpoint is hypophosphatasia. Hypophosphatasia is a bone disease with congenital metabolic disorders, with a structural gene mutation in TNAP that causes the AP activity in the blood to be lower than normal, manifesting as bone dysplasia. To date, more than 200 TNAP mutant genotypes have been reported. Although not yet curable, the success of recent mesenchymal stem cell transplantation has provided new ideas for the treatment of the disease. In addition, in the tissues that transport nutrients, such as the surface of the small intestine epithelium brush border, the surface of the fetal syncytium trophoblast, the surface membrane of the bile canal surface, ALP content is high, suggesting that ALP is related to the transfer and metabolism of phosphoric acid. In conclusion, ALPs in different tissues and organs have different physiological functions, but some are still unknown.

Applications

ALP has a wide range of uses in medicine and molecular biology. In clinical medicine, determination of serum ALP activity has become an important means for diagnosing and monitoring various diseases, including obstructive jaundice, primary liver cancer, secondary liver cancer, and cholestatic hepatitis. A significant elevation of blood-intestinal ALP can be seen in various intestinal diseases. ALP isoenzymes have gradually become known as a marker of tumor tissue. Bone ALP as a marker of abnormal bone metabolism has received more and more clinical attention. In animal feeding and disease diagnosis, ALP is an important biochemical indicator of reactive osteoblast activity, bone formation status, and calcium and phosphorus metabolism. For immunological research, ALP-labeled antibodies have been widely used for enzyme-linked immunofluorescence (ELISA) and Western blot analysis. Interaction of ALP with a developer or dephosphorylated substrate that emits light reveals the presence of target and detection complexes. In biochemistry and molecular biology, the use of ALP to catalyze the removal of the 5’ terminal phosphate group of a DNA molecule to prevent vector self-ligation is one of the conventional means in gene cloning.

Reference

  1. Holtz K M, Kantrowitz E R. The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site-specific mutagenesis. Febs Letters, 1999, 462(1-2):7–11.

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