Biochemistry - The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

Types of reactions catalyzed by enzymes
Substitution at the phosphorus atom
Phosphatases

Nucleophilic Substitution at the Phosphorus Atom (Table 7-1, reaction type 1.B) represents a critically important Class of reactions due to the vast number of Enzymes involved and their central role in METABOLISM/26.html">Energy Metabolism.

Phosphatases catalyze the Hydrolysis of phosphoric esters to yield inorganic phosphate:

or, in abbreviated form,

Two groups of enzymes, acidic and alkaline phosphatases, are non-specific enzymes capable of cleaving A wide variety of phosphoric acid esters. Other phosphatases, such as glucose-6-phosphatase and fructose-1,6-diphosphatase, exhibit strict substrate Specificity toward a single substrate. The physiological function of non-specific phosphatases remains unclear. Alkaline phosphatases are found in Bacteria, Fungi, and the Tissues of higher animals, but are absent in higher plants. In E. coli Cells, alkaline phosphatase is located predominantly in the periplasmic space. This enzyme has been detected in the brush border of renal tubular epithelium, intestinal epithelial cells, osteocytes, and osteoblasts. Alkaline phosphatase is almost completely absent in erythrocytes, Muscles, and other tissues not actively involved in nutrient transport. According to one theory, this enzyme ensures the generation of inorganic phosphate wherever a cellular demand arises. E. coli alkaline phosphatase is a dimer with a Molecular Weight of ~89,000, requires Zn²⁺ ions, is allosterically activated by Mg²⁺, and has an optimum pH above 8 [49]. Incubation of the enzyme with inorganic phosphate at pH ~4 leads to The formation of a phosphoenzyme. Experiments using ³²P-labeled phosphate established that the phosphate group attaches to a reactive Serine residue within the sequence

The exact same sequence has been found in mammalian alkaline phosphatases, which also exhibit a similar requirement for Metal Ions [50]. This sequence is analogous to the Amino Acid Sequence in the active sites of Serine proteinases, with the sole difference that Alanine replaces the Glycine residue in alkaline phosphatases. It is possible that these two groups of enzymes evolved from a common ancestral protein [51].

A characteristic feature of alkaline phosphatase from E. coli is that only one of the active sites Functions at any given moment. Based on this catalytic behavior, it has been proposed [52] that cooperative subunit interactions lead to an alternating change in the catalytic efficiency of the active sites via a flip-flop mechanism (see Chapter 8, Section 3.3). However, this hypothesis has been called into question [53].

Acid phosphatases have a pH optimum of ~5 and are inhibited by fluoride ions. They occur in both PLANT AND ANIMAL tissues. In bone, high concentrations of acid phosphatases are found in osteoclasts, whose function is to resorb calcium from Bone tissue. A phosphoenzyme has been isolated for both plant and animal alkaline phosphatases [54, 55]. Upon short-term incubation of the enzyme with ³²P-labeled p-nitrophenyl phosphate followed by exposure to an alkaline denaturing environment, a covalently labeled protein was obtained. Subsequent alkaline hydrolysis of this protein yielded ³²P-labeled N₁-phosphohistidine:

Pyrophosphatases, present in all cells, catalyze the hydrolysis of inorganic pyrophosphate to form two molecules of Pi (Supplement 3-A). The highly active E. coli pyrophosphatase has a turnover number exceeding 2∙10^-4 s^-1 at 37°C. To rapidly hydrolyze the pyrophosphate generated by bacterial metabolism, as few as 1,000 enzyme molecules per Cell are sufficient [56].