Biochemical studies of the enzyme have focused on proteins of ADSL from nonhuman species, the ADSL structure from the crystallized protein of Thermotoga maritime has been used, along with DNA sequencing data, to construct homology models for a variety of other organisms, including human ADSL. A variety of studies have been done using the equivalent enzyme from Bacillus subtilis, which shares a significant percentage of identity along with about some percentage of similarity in amino acid sequence with the human enzyme. Homology models overlaid on each other show a high degree of overlap between the enzymes.The family of enzymes to which ADSL belongs and that catalyze β-eliminations in which fumarate is one of the products are homotetramers with four active sites composed of amino acid residues from three distinct subunits. Much is known about the active site of human ADSL due to studies of the active site in the B. subtilis ADSL through affinity labeling and site-directed mutagenesis. While there is some variability among species in the sequencing of ADSL, the active site of the enzyme contains many residues that are conserved across species and have been shown to be critical to the enzymes function. His68 and His141 seem to serve as the general acid and base catalysts, and are critical to the catalyzing reaction of the substrate. His89 seems to enhance the binding of the substrates phosphoryl group and orient adenylosuccinate for catalysis. All three histidines are conserved throughout the 28 species for which the structure of ADSL is known.
Mechanism
In cell biology, actinomycin D is shown to have the ability to inhibit transcription. Actinomycin D does this by binding DNA at the transcription initiation complex and preventing elongation of RNA chain by RNA polymerase. History
Actinomycin D was the first antibiotic shown to have anti-cancer activity. It was first isolated by Selman Waksman and his co-worker H. Boyd Woodruff in 1940. It was approved by the U.S. Food and Drug Administration (FDA) on December 10, 1964, and launched by Merck Sharp and Dohme under the trade name Cosmegen. Research use
Because actinomycin can bind DNA duplexes, it can also interfere with DNA replication, although other chemicals such as hydroxyurea are better suited for use in the laboratory as inhibitors of DNA synthesis. Actinomycin D and its fluorescent derivative, 7-aminoactinomycin D (7-AAD), are used as stains in microscopy and flow cytometry applications. The affinity of these stains/compounds for GC-rich regions of DNA strands makes them excellent markers for DNA. 7-AAD binds to single stranded DNA; therefore it is a useful tool in determining apoptosis and distinguishing between dead cells and live ones. References
External links
"Dactinomycin". Drug Information Portal. U.S. National Library of Medicine.