Site-specific deoxyribonuclease

Site-specific deoxyribonuclease (type 11)— restriction endonuclease ... 

Type II site-specific deoxyribonuclease [EC 3.1.21.4], also referred to as type II restriction enzyme, catalyzes the endonucleolytic cleavage of DNA to give specific, double-stranded fragments with terminal 5 -phosphates. Magnesium ions are required as cofactors. 

Protein enzymes have been classified into six major classes according to the nature of the reaction they catalyze. Enzyme Commission (EC) numbers have been assigned to all enzymes by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Generally, each enzyme has a unique number, but there are exceptions—the restriction enzymes which cut nucleic acids are one such exceptional group. The official name and munber for this group of enzymes (and there are thousands of them) is Type II site-specific deoxyribonuclease, EC 3.1.21.4. Many other details of enzymes and their kinetics are given in Chap. 5. 

Enzymes catalyzing cleavage of DNA, including endo-deoxyribonucleases that generate 5 -phosphomono-esters [EC 3.1.21.x], endodeoxyribonucleases that produce products other than 5 -phosphomonoesters [EC 3.1.22.x], site-specific endodeoxyribonucleases acting on altered bases [EC 3.1.25.x], and exodeoxyribonucleases producing 5 -phosphomonoesters [EC 3.1.11.x]. A few examples are ... 

Two examples where metal ions confer stability or increased activity in proteins are human deoxyribonuclease (rhDNase, Pulmozyme ), and Factor VIII. In the case of rhDNase, Ca2+ ions (up to 100 mM) increased the stability of the enzyme through a specific binding site (64). In fact, the removal of calcium ions from the solution with EGTA caused an increase in deamidation and aggregation. However, this effect was observed only with Ca+2 ions other divalent cations, Mg2+, Mn2+, and Zn2+, were observed to destabilize rhDNase. Similar effects were observed in Factor VIII. Ca2+ and Sr2+ ions stabilized the protein, whereas others such as Mg2+, Mn2+ and Zn2+, Cu2+, and Fe2+ destabilized the enzyme (65). In a separate study with Factor VIII, a significant increase in the aggregation rate was observed in the presence of Al3+ ions (66). The authors note that other excipients like buffer salts are often contaminated with Al3+ ions and illustrate the need to use excipients of appropriate quality in formulated products. 

In the body, enzymes are compartmentalized and function under highly restricted conditions. Some enzymes (e.g., proteinases) are not substrate-specific. When present in active form in an inappropriate part of the body, they act indiscriminately and cause considerable damage to the tissue. Inhibitors inactivate these enzymes at sites where their action is not desired. Proteinase inhibitors, which are themselves proteins, are widely distributed in intracellular and extracellular fluids. Protein inhibitors of enzymes other than proteinases are relatively rare. Such inhibitors are available for a-amylases, deoxyribonuclease I, phospholipase A, and protein kinases. 

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