Alkaline phosphatases (ALPs) are a superfamily of metalloenzymes that are widely found in organisms ranging from bacteria to human and catalyze the hydrolytic removal of phosphate from a variety of molecules. Alkaline phosphatase is found in all three major biological kingdoms and several have been isolated and characterized. These enzymes have been identified and characterized from many eukaryotic and prokaryotic sources and comprise several distinct subgroups based upon substrate specificity, molecular weight, and sensitivity to known inhibitors. There are three major ALP isoenzymes: intestinal, placental, and liver/bone/kidney (tissue non-specific alkaline phosphatase isoenzyme, TNAP).
Study of Alkaline phosphatases
Although phosphatase activity was first reported in the early 1900s, and the term alkaline phosphatase was first coined in the 1930s, the properties of alkaline phosphatases were not the subject of intense research studies until the 1950s and 1960s. During this period, alkaline phosphatases was purified and kinetically characterized from a number of sources and generally described as a dimeric metalloenzyme, which non-specifically catalyzes the hydrolysis of phosphomonoesters to yield phosphate and the corresponding alcohol. Morton studied the transphosphorylation activity of alkaline phosphatase, or the ability of alkaline phosphatase to transfer phosphate from the substrate to an alcohol, resulting in the formation of another phosphomonoester.
In research completed around 1960, alkaline phosphatase with a 32P-labeled phosphoserine was isolated from preparations of alkaline phosphatases incubated with radioactive inorganic phosphate. From these studies it was proposed that catalysis proceeded through a serine phosphate intermediate. Another area of study in the 1960s was the intragenic complementation found to exist in E. coli alkaline phosphatase. In these studies it was found in certain cases that if the alkaline phosphatases subunits from two E. coli strains producing different inactive versions of alkaline phosphatases were combined to form an alkaline phosphatases hybrid, the resulting holoenzyme would have some activity restored. Research in all of these aforementioned areas has continued to the present day.
Alkaline phosphatase is encoded by the phoA gene, and first ligated into a plasmid and mapped with restriction enzymes by Inouye and coworkers. Derivatives of this plasmid have been used as an alkaline phosphatase source in many laboratories, expressed in an E. coli strain which lacks the phoA gene on the chromosome. These constructs increased the protein yield of alkaline phosphatase without cross contamination of the native alkaline phosphatase from the E. coli host. Hence the construction and ex
At about the same time, the amino acid sequence of the protein and nucleic acid sequence of the gene were determined separately and compared. From these experiments it was determined that alkaline phosphatase was modified after transcription by cleavage of an N-terminal peptide signal sequence consisting of amino acids after secretion into the periplasmic space.
Multiple structural studies, which mostly occurred in the 1980s and the 1990s, were used to characterize the enzyme structurally and used to propose a catalytic mechanism for alkaline phosphatase. High-resolution crystal structures of alkaline phosphatase from E. coli, shrimp and human placenta can currently be found in the Protein Data Bank. From the E. coli alkaline phosphatase structure, the first deposited, a mechanism based on two metal ion catalysis was proposed.
Function of Alkaline phosphatases
Alkaline phosphatase has been proposed to be both a transporter and provider of inorganic phosphate in bacterial cells. A common environment for E. coli is one with only trace phosphate levels. Alkaline phosphatase is one of 90 proteins that are translated under phosphate limiting conditions. For instance, Porin E, a protein in the outer membrane of E. coli, is responsible for transporting phosphorylated compounds from the surroundings into the periplasm, and alkaline phosphatase catalyzes the hydrolysis of the wide variety of phosphomonoesters that enter the cell through Porin E. In turn, the liberated phosphate is bound and transported to the cytosol by an assembly of several proteins known as the phosphate-specific transport system complex (Pst), which spans the inner membrane.
Research investigating the different functions of the several isoforms of alkaline phosphatase expressed in human tissues has helped to provide information about the different roles alkaline phosphatase has in mammals.
The physiological function of ALP is largely unknown; however, it has been shown that ALP can detoxify Gram-negative lipopolysaccharide (LPS) by removing terminal phosphate groups. The TNAP plays an important role in matrix mineralization. It has been proposed that the activity of TNAP is required to generate the inorganic phosphate needed for hydroxyapatite crystallization, and ATP and PPi have been explored as potential substrates. However, the ability of TNAP to hydrolyze PPi has been hypothesized to be important to promote calcification by hydrolyzing this potent inhibitor of mineralization. The TNAP isozyme has been proposed to play an important role in skeletal mineralization. Hypophosphatasia is an inherited disorder of osteogenesis that is characterized by deactivating mutations in the tissue non-specific alkaline phosphatase gene. The severity of hypophosphatasia can range from brittle bones in adulthood to prenatal death due to a lack of a mineralized skeleton. Moreover, partial reduction of TNAP activity and impairment of osteoblast maturation is observed in osteoblasts differentiated in vitro from precursor cells prepared from Znt5–/– mice.
The increased ex