User:R. Jeremy Johnson/RNaseA: Difference between revisions
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== '''Introduction''' == | == '''Introduction''' == | ||
<Structure load='7RSA' size='300' frame='true' align='left' caption='RNase A' scene='Sandbox_Reserved_192/Second_rnase_structure_blue/1' /> | <Structure load='7RSA' size='300' frame='true' align='left' caption='RNase A' scene='Sandbox_Reserved_192/Second_rnase_structure_blue/1' /> | ||
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=='''Structure'''== | =='''Structure'''== | ||
RNase A is made up of a single polypeptide chain of 124 residues. Of the 20 natural amino acids, RNase A possesses 19 of them, excluding tryptophan. This single polypeptide chain is cross-linked internally by four | RNase A is made up of a single polypeptide chain of 124 residues. Of the 20 natural amino acids, RNase A possesses 19 of them, excluding tryptophan. This single polypeptide chain is cross-linked internally by four disulfide linkages, which contribute to the stability of RNase A. Long four-stranded anti-parallel <scene name='Sandbox_Reserved_192/Beta_sheet/4'>ß-sheets</scene> and three short <scene name='Sandbox_Reserved_192/Alpha_helices/2'>α-helices</scene> make up the <scene name='Sandbox_Reserved_192/Secondary_structure/3'>secondary structure</scene> of RNase A (Raines). The amino acid sequence was discovered to determine the three-dimensional structure of RNase A by Christian Anfinsen in the 1950s. Urea was used to denature RNase A, and mercaptoethanol was used to reduce and cleave the four disulfide bonds in RNase A to yield eight Cys residues. Catalytic activity was lost due to denaturation. When the urea and mercaptoethanol were removed, the denatured ribonuclease refolded spontaneously into its correct tertiary structure with restoration of its catalytic activity. Disulfide bonds were also reformed in the same position. The Anfinsen experiment provided evidence that the amino acid sequence contained all the information required for the protein to fold into its native three-dimensional structure. Anfinsen received the 1972 Nobel Prize in Chemistry for his work with RNase A. Nevertheless, ensuing work showed some proteins require further assistance, such as molecular chaperones, to fold into their native structure. | ||
The amino acid sequence was discovered to determine the three-dimensional structure of RNase A by Christian Anfinsen in the 1950s. Urea was used to denature RNase A, and mercaptoethanol was used to reduce and cleave the four disulfide bonds in RNase A to yield eight Cys residues. Catalytic activity was lost due to denaturation. When the urea and mercaptoethanol were removed, the denatured ribonuclease refolded spontaneously into its correct tertiary structure with restoration of its catalytic activity. Disulfide bonds were also reformed in the same position. The Anfinsen experiment provided evidence that the amino acid sequence contained all the information required for the protein to fold into its native three-dimensional structure. Anfinsen received the 1972 Nobel Prize in Chemistry for his work with RNase A. Nevertheless, ensuing work showed some proteins require further assistance, such as molecular chaperones, to fold into their native structure. | |||
=Ribonuclease A Catalysis= | =Ribonuclease A Catalysis= | ||
[[Image:RNaseAIII.png|300px|left|thumb|Figure I: Bovine Ribonuclease A. Colored residues are representative of amino acids important to both the acid base catalysis (Red: His12 and 119) and stabilization of the transition state (Blue: Lys41). Figure generated via ''Pymol'' ]] | [[Image:RNaseAIII.png|300px|left|thumb|Figure I: Bovine Ribonuclease A. Colored residues are representative of amino acids important to both the acid base catalysis (Red: His12 and 119) and stabilization of the transition state (Blue: Lys41). Figure generated via ''Pymol'' ]] | ||
{{STRUCTURE_7rsa | PDB=7RSA | SCENE= Sandbox_Reserved_193/Rnasei_a/1 }} | {{STRUCTURE_7rsa | PDB=7RSA | SCENE= Sandbox_Reserved_193/Rnasei_a/1 }} | ||
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Acid/base catalysis by an enzyme is dependent on the pH of the environment and the pKa's of their residues. The pKa value will increase for an acidic residue if the environment is hydrophobic or if the adjacent residues are of similar charges. In the same environmental conditions, a basic residue will decrease the pKa. This ability to alter the pKa of certain residues such as histidines, increases the diversity of reactions that an enzyme can perform. | Acid/base catalysis by an enzyme is dependent on the pH of the environment and the pKa's of their residues. The pKa value will increase for an acidic residue if the environment is hydrophobic or if the adjacent residues are of similar charges. In the same environmental conditions, a basic residue will decrease the pKa. This ability to alter the pKa of certain residues such as histidines, increases the diversity of reactions that an enzyme can perform. | ||
==Active Site Structure== | ==Active Site Structure== | ||
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Early studies on RNase A catalysis showed that alkylation of His12 and His119 significantly decreased its catalytic activity, prompting the hypothesis that these two histidines were the acid/base catalyst. Confirmation of this hypothesis came when these histidines were replaced with alanine and the reaction rates of either mutation dropped by ten-thousand fold <ref name="Wlodrawer1988" />. | Early studies on RNase A catalysis showed that alkylation of His12 and His119 significantly decreased its catalytic activity, prompting the hypothesis that these two histidines were the acid/base catalyst. Confirmation of this hypothesis came when these histidines were replaced with alanine and the reaction rates of either mutation dropped by ten-thousand fold <ref name="Wlodrawer1988" />. | ||
=='''Acid Base Catalysis by RNase A'''== | |||
RNase A catalyzes the cleavage of the Phosphodiester bonds in two steps: the formation of the pentavalent phosphate transition state and subsequent degradation of the 2’3’ cyclic phosphate intermediate. An important part of the reaction is the ability of histidine (His 12 and His119) to both accept and donate electrons, allowing these histidine to be an acid or a base, making the reaction pH dependent <ref name="Raines1998" />. | RNase A catalyzes the cleavage of the Phosphodiester bonds in two steps: the formation of the pentavalent phosphate transition state and subsequent degradation of the 2’3’ cyclic phosphate intermediate. An important part of the reaction is the ability of histidine (His 12 and His119) to both accept and donate electrons, allowing these histidine to be an acid or a base, making the reaction pH dependent <ref name="Raines1998" />. | ||
[[Image:MechIII.png|450px|left|thumb|Figure II: RNase A Catalysis. (A) Initial attack of 2'hydroxyl stabilized by His12. (B) Pentavalent phosphorous intermediate. (C) 2'3' cyclic intermediate degradation. (D) Finished products: Two distinctive nucleotide sequences. Figure generated via ''Chemdraw'']] | [[Image:MechIII.png|450px|left|thumb|Figure II: RNase A Catalysis. (A) Initial attack of 2'hydroxyl stabilized by His12. (B) Pentavalent phosphorous intermediate. (C) 2'3' cyclic intermediate degradation. (D) Finished products: Two distinctive nucleotide sequences. Figure generated via ''Chemdraw'']] | ||
RNA hydrolysis begins when <scene name='Sandbox_Reserved_193/His12a_a/1'>His12</scene> abstracts a proton from the 2’ OH group on RNA; thus, assisting in the nucleophilic attack of the 2’ oxygen on the electrophilic phosphorus atom. A transition state is then formed, having a pentavalent phosphate, which is stabilized by the positively charged amino group of <scene name='Sandbox_Reserved_193/Lys41a_a/1'>Lys41</scene> and the main chain amide nitrogen of Phe120. <scene name='Sandbox_Reserved_193/His119a_a/1'>His119</scene> then protonates the 5' oxygen on the ribose ring and the transition state falls to form a 2’3’cyclic phosphate intermediate <ref name="Raines1998" />. | RNA hydrolysis begins when <scene name='Sandbox_Reserved_193/His12a_a/1'>His12</scene> abstracts a proton from the 2’ OH group on RNA; thus, assisting in the nucleophilic attack of the 2’ oxygen on the electrophilic phosphorus atom. A transition state is then formed, having a pentavalent phosphate, which is stabilized by the positively charged amino group of <scene name='Sandbox_Reserved_193/Lys41a_a/1'>Lys41</scene> and the main chain amide nitrogen of Phe120. <scene name='Sandbox_Reserved_193/His119a_a/1'>His119</scene> then protonates the 5' oxygen on the ribose ring and the transition state falls to form a 2’3’cyclic phosphate intermediate <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" />. | |||
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''' = | ||
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Further binding pocket characterization was performed using <scene name='Sandbox_Reserved_194/1rcn_structure/3'>RNase A complexed with the oligonucleotide d(ApTpApApG)</scene> ([[1rcn]]). This <scene name='Sandbox_Reserved_194/1rcn_just_dtda/3'>tetramer</scene> was important in determining the specificity of the binding sites of RNase A. In this complex, the B1 site is thought to exclusively bind to pyrimidine bases due to steric interactions and <scene name='Sandbox_Reserved_194/1rcn_thr45/1'>hydrogen bonding to Thr45d</scene>. When Thr45 was mutated to glycine, purines readily bound to the B1 site. ‘<ref>PMID: 8193116</ref>’ This interaction appears to be the driving force behind its inability to bind purines. While binding of other nucleobases to the B2 and B3 sites is possible, the imaging of this complex elucidated the preferences for adenosine bases at these two positions. In addition to its catalytic activity His119 has also been shown to be important in substrate specificity. This is due to the <scene name='Sandbox_Reserved_194/1rcn_his/10'>pi stacking between His119 and A3 </scene>. When this site was mutated, the affinity for a poly(A) substrate was decreased by 104-fold. ‘<ref>PMID: 21391696</ref>It also establishes hydrogen bonding between <scene name='Sandbox_Reserved_194/1rcn_hydrogen_bonding/10'>Asn71-A3, Gln69-A3 and Gln69-A4</scene>. ‘<ref>PMID:8063789</ref>’ | Further binding pocket characterization was performed using <scene name='Sandbox_Reserved_194/1rcn_structure/3'>RNase A complexed with the oligonucleotide d(ApTpApApG)</scene> ([[1rcn]]). This <scene name='Sandbox_Reserved_194/1rcn_just_dtda/3'>tetramer</scene> was important in determining the specificity of the binding sites of RNase A. In this complex, the B1 site is thought to exclusively bind to pyrimidine bases due to steric interactions and <scene name='Sandbox_Reserved_194/1rcn_thr45/1'>hydrogen bonding to Thr45d</scene>. When Thr45 was mutated to glycine, purines readily bound to the B1 site. ‘<ref>PMID: 8193116</ref>’ This interaction appears to be the driving force behind its inability to bind purines. While binding of other nucleobases to the B2 and B3 sites is possible, the imaging of this complex elucidated the preferences for adenosine bases at these two positions. In addition to its catalytic activity His119 has also been shown to be important in substrate specificity. This is due to the <scene name='Sandbox_Reserved_194/1rcn_his/10'>pi stacking between His119 and A3 </scene>. When this site was mutated, the affinity for a poly(A) substrate was decreased by 104-fold. ‘<ref>PMID: 21391696</ref>It also establishes hydrogen bonding between <scene name='Sandbox_Reserved_194/1rcn_hydrogen_bonding/10'>Asn71-A3, Gln69-A3 and Gln69-A4</scene>. ‘<ref>PMID:8063789</ref>’ | ||
From the studies on RNase A complexes with deoxy nucleic acid tetramers, it has been established that this enzyme recognizes the substrate on both its phosphate backbone and on individual nucleobases. RNase A has a nonspecific B0 site, a B1 site specific to pyrimidines and a B3 and B4 site with a preference for adenosine bases. Similar to other enzymes, RNase A uses the hydrogen bonding distance between amino acids and the substrate to bind specifically to certain nucleobases. Studying the substrate recognition and specificity of enzymes such as RNase A is an important step in understanding the regulation of RNA within biological systems. | |||
="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|300px|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'']] | |||
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" /> | |||