Alkaline phosphatase

The enzyme alkaline phosphatase (ALP, alkaline phenyl phosphatase, also abbreviated PhoA) is a phosphatase with the physiological role of dephosphorylating compounds. The enzyme is found across a multitude of organisms, prokaryotes and eukaryotes alike, with the same general function, but in different structural forms suitable to the environment they function in. Alkaline phosphatase is found in the periplasmic space of E. coli bacteria. This enzyme is heat stable and has its maximum activity at high pH. In humans, it is found in many forms depending on its origin within the body – it plays an integral role in metabolism within the liver and development within the skeleton. Due to its widespread prevalence in these areas, its concentration in the bloodstream is used by diagnosticians as a biomarker in helping determine diagnoses such as hepatitis or osteomalacia.

The level of alkaline phosphatase in the blood is checked through the ALP test, which is often part of routine blood tests. The levels of this enzyme in the blood depend on factors such as age, sex, or blood type. Blood levels of alkaline phosphatase also increase by two to four times during pregnancy. This is a result of additional alkaline phosphatase produced by the placenta and the liver. Additionally, abnormal levels of alkaline phosphatase in the blood could indicate issues relating to the liver, gall bladder or bones. Kidney tumors and infections as well as malnutrition have also shown abnormal level of alkaline phosphatase in blood. Alkaline phosphatase levels in a cell can be measured through a process called the "scoring method". A blood smear is usually taken and stained to categorize each leukocyte into specific leukocyte alkaline phosphatase indices. This marker is designed to distinguish leukocytes and determine different enzyme activity from each sample's extent of staining.

In gram-negative bacteria, such as Escherichia coli, alkaline phosphatase is located in the periplasmic space, external to the inner cell membrane and within the peptidoglycan portion of the cell wall. Since the periplasmic gap is more prone to environmental variation than the inner cell, alkaline phosphatase is suitably resistant to inactivation, denaturation, or degradation. This characteristic of the enzyme is uncommon to many other proteins.

The precise structure and function of the isozyme in E. coli is solely geared to supply a source of inorganic phosphate when the environment lacks this metabolite. The inorganic phosphates are then bound to carrier proteins which deliver the inorganic phosphates to a specific high-affinity transport system, known as the phosphate-specific transport system, which transports phosphate across the cytoplasmic membrane.

While the outer membrane of E. coli contains porins that are permeable to phosphorylated compounds, the inner membrane does not. An issue arises in how to transport such compounds across the inner membrane and into the cytosol. The strong anionic charge of phosphate groups along with the remainder of the compound make phosphorylated compounds very much immiscible in the nonpolar region of the bilayer. The solution arises in cleaving the phosphate group away from the compound via ALP. for translocation into the cytosol, The main purpose of dephosphorylation by alkaline phosphatase is to increase the rate of diffusion of phosphorylated molecules into the cells while inhibiting them from diffusing out.

Alkaline phosphatase is a zinc-containing dimeric enzyme with the MW: 86,000 Da, each subunit containing 429 amino acids with four cysteine residues linking the two subunits. Alkaline phosphatase contains four Zn ions and two Mg ions, with Zn occupying active sites A and B, and Mg occupying site C, so the fully active native alkaline phosphatase is referred to as (Zn_AZn_BMg_C)₂ enzyme. The mechanism of action of alkaline phosphatase involves the geometric coordination of the substrate between the Zn ions in the active sites.

Alkaline phosphatase in E. coli is uncommonly soluble and active within elevated temperature conditions such as 80 °C. Due to the kinetic energy induced by this temperature the weak hydrogen bonds and hydrophobic interactions of common proteins become degraded and therefore coalesce and precipitate. However, upon dimerization of alkaline phosphatase, the bonds maintaining its secondary and tertiary structures are effectively buried such that they are not affected as much at this temperature. Furthermore, even at more elevated temperatures such as 90 °C alkaline phosphatase has the unusual characteristic of reverse denaturation. Due to this, although it ultimately denatures at about 90 °C it has the added ability to accurately reform its bonds and return to its original structure and function once cooled back down.

Alkaline phosphatase in E. coli is located in the periplasmic space and can thus be released using techniques that weaken the cell wall and release the protein. Due to the location of the enzyme, and the protein layout of the enzyme, the enzyme is in solution with a smaller amount of proteins than there are in another portion of the cell. The proteins' heat stability can also be taken advantage of when isolating this enzyme (through heat denaturation). In addition, alkaline phosphatase can be assayed using p-nitrophenyl phosphate. A reaction where alkaline phosphatase dephosphorylates the non-specific substrate, p-nitrophenyl phosphate in order to produce p-nitrophenol (PNP) and inorganic phosphate. PNP's yellow color, and its λ_max at 410 allows spectrophotometry to determine enzymatic activity. Some complexities of bacterial regulation and metabolism suggest that other, more subtle, purposes for the enzyme may also play a role for the cell. In the laboratory, however, mutant Escherichia coli lacking alkaline phosphatase survive quite well, as do mutants unable to shut off alkaline phosphatase production.