User:R. Jeremy Johnson/RNaseA: Difference between revisions
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In a secondary and separate reaction, the 2’,3’ cyclic phosphate is hydrolyzed to a mixture of 2'phosphate and 3' hydroxyl. His12 donates a proton to the leaving group of this reaction, the 3’ oxygen of the cyclic intermediate. Simultaneously, His-119 abstracts the proton from a water molecule, activating it for nucleophilic attack. The activated water molecule attacks the cyclic phosphate causing the cleavage of the 2'3’ cyclic phosphate intermediate. The truncated nucleotide is then released with a 3’ phosphate group <ref name="Raines1998" />. | In a secondary and separate reaction, the 2’,3’ cyclic phosphate is hydrolyzed to a mixture of 2'phosphate and 3' hydroxyl. His12 donates a proton to the leaving group of this reaction, the 3’ oxygen of the cyclic intermediate. Simultaneously, His-119 abstracts the proton from a water molecule, activating it for nucleophilic attack. The activated water molecule attacks the cyclic phosphate causing the cleavage of the 2'3’ cyclic phosphate intermediate. The truncated nucleotide is then released with a 3’ phosphate group <ref name="Raines1998" />. | ||
= '''Ribonuclease A Substrate Binding''' = | = '''Ribonuclease A Substrate Binding''' = | ||
<Structure load='1RTA' size=' | <Structure load='1RTA' size='350' frame='true' align='right' caption='Ribonuclease A complexed with thymidylic acid tetramer and ApTpApApG showing pi stacking and hydrogen bonding' scene='Sandbox_Reserved_194/1rta_structure/2'/> | ||
RNase A is an endonuclease that cleaves and breaks down RNA using acid base catalysis. RNase A has been a model protein for studies on the stability, folding and chemistry of proteins. ‘<ref>PMID:11848924</ref>’ It is also essential in protein regulation within the body due to its function of RNA degradation. | RNase A is an endonuclease that cleaves and breaks down RNA using acid base catalysis. RNase A has been a model protein for studies on the stability, folding and chemistry of proteins. ‘<ref>PMID:11848924</ref>’ It is also essential in protein regulation within the body due to its function of RNA degradation. | ||
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='''Inhibitors'''= | ='''Inhibitors'''= | ||
Due to the highly catalytic nature of RNase A for RNA strands, mammalian cells have developed a protective inhibitor to prevent pancreatic ribonucleases from degrading cystolic RNA. Ribonuclease Inhibitor (RI) tightly associates to the active site due to its non-globular nature. RI is a large 50 kD protein that is composed of 16 repeating alpha and beta chains, giving it a noticable horseshoe like appearance. It has been suggested that RI has the highest protein-protein interactions with an approximate dissociation constant (Kd) of 5.8 X 10-14 for almost all types of RNases <ref name="Vicentini1996" />. | [[Image:RI.PNG|280px|Right|thumb|Figure III: Ribonuclease Inhibitor-RNase A Complex. Left, Ribonuclease Inhibitor (RI)is composed of alternating alpha helix (blue) and beta sheets (green). Right, RI-RNase A inhibition forms when RI complex with the active site cleft of RNase (yellow). Figure generated via ''Pymol'']] Due to the highly catalytic nature of RNase A for RNA strands, mammalian cells have developed a protective inhibitor to prevent pancreatic ribonucleases from degrading cystolic RNA. Ribonuclease Inhibitor (RI) tightly associates to the active site due to its non-globular nature. RI is a large 50 kD protein that is composed of 16 repeating alpha and beta chains, giving it a noticable horseshoe like appearance. It has been suggested that RI has the highest protein-protein interactions with an approximate dissociation constant (Kd) of 5.8 X 10-14 for almost all types of RNases <ref name="Vicentini1996" />. The ability to be selective for almost all types of RNases, and yet retain such a high Kd is product of its mechanism. The interior residues of the horseshoe shaped RI are able to bind to the charged residues of the active site cleft of RNase A, such as Lys7, Lys9, Lys 41 and Gln11. By studying the amphibian RNase, Onconase, the residues Lys7 and Gln11 of RNase A were shown to be the most important in this interaction. In onconase, these residues are replaced with non-charged amino acids, which help prevent the binding of RI to the protein <ref name="Turcotte2008" /> | ||
The ability to be selective for almost all types of RNases, and yet retain such a high Kd is product of its mechanism. The interior residues of the horseshoe shaped RI are able to bind to the charged residues of the active site cleft of RNase A, such as Lys7, Lys9, Lys 41 and Gln11. By studying the amphibian RNase, Onconase, the residues Lys7 and Gln11 of RNase A were shown to be the most important in this interaction. In onconase, these residues are replaced with non-charged amino acids, which help prevent the binding of RI to the protein <ref name="Turcotte2008" /> | |||