Deoxyribonuclease active site

P. A. Price, S. Moore, and W- H. Stein. Alkylation of a histidine residue at the active site of bovine pancreatic deoxyribonuclease. J. Biot. Chem. 244 924-928... 

A kinetic study on the activation of deoxyribonuclease I by magnesium has shown that the activation curve is biphasic (the substrate being the Mg salt of bovine spleen DNA). This indicates that activation occurs at two sites on the protein. Free Mg + was required for enzyme activity, confirming that a metallo-enzyme as well as a metallo-substrate is necessary for deoxyribonuclease I activity. In contrast, it has been shown that the pyrophosphatase activity of bovine brain alkaline phosphatase depends on Mg + bound to the enzyme but not on the formation of a magnesium-substrate complex. 

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. 

Fig. 5. The execution phase of apoptosis. An apoptotic stimulus causes the release of cytochrome c from mitochondria. The first box contains the components required to activate caspase 9 card refers to the caspase-recruitment domain. Caspase 9 then activates caspase 3 (second box), which in turn activates caspase 6 (last box). The amino-acid sequences at the cleavage sites are shown. Caspases 3 and 6 also have a prodomain that is not present in the active protease in caspase 6 an additional cleavage removes a small portion of the middle of the protein. Various substrates of the caspases are shown, including the pathway by which caspase-activated deoxyribonuclease (CAD) is activated which in turn leads to DNA...

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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