RNase A - Revision history

Alexander Berchansky at 11:55, 27 February 2019

Alexander Berchansky — Wed, 27 Feb 2019 11:55:47 GMT

←Older revision		Revision as of 11:55, 27 February 2019
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	[[Image:1RCNnew.png\|thumb\|left\|200px\|ApTpApApG complexed with ribonuclease A]]		[[Image:1RCNnew.png\|thumb\|left\|200px\|ApTpApApG complexed with ribonuclease A]]
	{{Clear}}		{{Clear}}
-	Further binding pocket characterization was performed using <scene name='44/449690/Cv/13'>RNase A complexed with the oligonucleotide d(ApTpApApG)</scene> ([[1rcn]]). This <scene name='44/449690/Cv/14'>tetramer</scene> is closely positioned with the catalytic residues, <scene name='44/449690/Cv/15'>His12, Lys41, and His119</scene> and 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='44/449690/Cv/16'>hydrogen bonding to Thr45</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='44/449690/Cv/17'>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='44/449690/Cv/18'>Asn71-A3, Gln69-A3 and Gln69-A4</scene>. <ref>PMID:8063789</ref>	+	Further binding pocket characterization was performed using <scene name='44/449690/Cv/13'>RNase A complexed with the oligonucleotide d(ApTpApApG)</scene> ([[1rcn]]). This <scene name='44/449690/Cv/14'>tetramer</scene> is closely positioned with the catalytic residues, <scene name='44/449690/Cv/15'>His12, Lys41, and His119</scene> and 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='44/449690/Cv/16'>hydrogen bonding to Thr45</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='44/449690/Cv/17'>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='44/449690/Cv/18'>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.		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.

Alexander Berchansky at 11:36, 27 February 2019

Alexander Berchansky — Wed, 27 Feb 2019 11:36:40 GMT

←Older revision		Revision as of 11:36, 27 February 2019
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	<scene name='User:R._Jeremy_Johnson/RNaseA/Ri_rnasea_simple/1'>inhibitor (RI) (tan) bound to RNase A (red)</scene> ([[1dfj]]).		<scene name='User:R._Jeremy_Johnson/RNaseA/Ri_rnasea_simple/1'>inhibitor (RI) (tan) bound to RNase A (red)</scene> ([[1dfj]]).

-	Due to the high rate of RNA hydrolysis by RNase A, mammalian cells have developed a protective inhibitor to prevent pancreatic ribonucleases from degrading cystolic RNA. Ribonuclease Inhibitor (RI) tightly associates to the active site of RNase A due to its <scene name='User:R._Jeremy_Johnson/RNaseA/Ri_simple/1'>non-globular nature</scene>. RI is a 50 kD protein that is composed of 16 repeating subunits of alpha helices and beta sheets, giving it a noticeable <scene name='User:R._Jeremy_Johnson/RNaseA/Ri_nonglobular/1'>horseshoe like appearance</scene>. The RI-RNase protein-protein interaction has the highest known affinity of any protein-protein interactions with an approximate dissociation constant (''K''d) of 5.8 X 10-14 for almost all types of ribonucleases.<ref>PMID:7877692</ref> The ability to be selective for almost all types of RNases, and yet retain such a high Kd is product of its mechanism of inhibition. 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 <scene name='~~User:R._Jeremy_Johnson~~/~~RNaseA~~/~~Ri_rnasea_lys7_gln11_lys41~~/1'>Lys7, Gln11, and Lys41 </scene>. 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>PMID:18930025</ref>	+	Due to the high rate of RNA hydrolysis by RNase A, mammalian cells have developed a protective inhibitor to prevent pancreatic ribonucleases from degrading cystolic RNA. Ribonuclease Inhibitor (RI) tightly associates to the active site of RNase A due to its <scene name='User:R._Jeremy_Johnson/RNaseA/Ri_simple/1'>non-globular nature</scene>. RI is a 50 kD protein that is composed of 16 repeating subunits of alpha helices and beta sheets, giving it a noticeable <scene name='User:R._Jeremy_Johnson/RNaseA/Ri_nonglobular/1'>horseshoe like appearance</scene>. The RI-RNase protein-protein interaction has the highest known affinity of any protein-protein interactions with an approximate dissociation constant (''K''d) of 5.8 X 10-14 for almost all types of ribonucleases.<ref>PMID:7877692</ref> The ability to be selective for almost all types of RNases, and yet retain such a high Kd is product of its mechanism of inhibition. 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 <scene name='44/449690/Cv/19'>Lys7, Gln11, and Lys41 </scene>. 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>PMID:18930025</ref>
	</StructureSection>		</StructureSection>
	__NOTOC__		__NOTOC__

Alexander Berchansky at 11:32, 27 February 2019

Alexander Berchansky — Wed, 27 Feb 2019 11:32:01 GMT

←Older revision		Revision as of 11:32, 27 February 2019
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	[[Image:1RCNnew.png\|thumb\|left\|200px\|ApTpApApG complexed with ribonuclease A]]		[[Image:1RCNnew.png\|thumb\|left\|200px\|ApTpApApG complexed with ribonuclease A]]
	{{Clear}}		{{Clear}}
-	Further binding pocket characterization was performed using <scene name='~~Sandbox_Reserved_194~~/~~1rcn_structure~~/~~3' target='0~~'>RNase A complexed with the oligonucleotide d(ApTpApApG)</scene> ([[1rcn]]). This <scene name='~~Sandbox_Reserved_194~~/~~1rcn_just_dtda~~/~~3' target='0~~'>tetramer</scene> is closely positioned with the catalytic residues, <scene name='~~Sandbox_Reserved_194~~/~~1rcn_structure_active_site~~/1'>His12, Lys41, and His119</scene> and 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' target='0~~'>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' target='0~~'>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='44/449690/Cv/13'>RNase A complexed with the oligonucleotide d(ApTpApApG)</scene> ([[1rcn]]). This <scene name='44/449690/Cv/14'>tetramer</scene> is closely positioned with the catalytic residues, <scene name='44/449690/Cv/15'>His12, Lys41, and His119</scene> and 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='44/449690/Cv/16'>hydrogen bonding to Thr45</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='44/449690/Cv/17'>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='44/449690/Cv/18'>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.		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.

Alexander Berchansky at 11:05, 27 February 2019

Alexander Berchansky — Wed, 27 Feb 2019 11:05:29 GMT

←Older revision		Revision as of 11:05, 27 February 2019
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	[[Image:1RTAnew.png\|thumb\|left\|200px\|Thymidylic acid tetramer complexed with ribonuclease A]]		[[Image:1RTAnew.png\|thumb\|left\|200px\|Thymidylic acid tetramer complexed with ribonuclease A]]
	{{Clear}}		{{Clear}}
-	To determine the structural characteristics of RNA substrate binding to RNase A, X-ray crystallography was used to image inhibitory DNA tetramers bound to RNase A. DNA lacks the 2’OH essential to RNA cleavage, making the complex more conducive to crystallography. <scene name='~~Sandbox_Reserved_194~~/~~1rta_structure~~/~~2' target='0~~'>The complex</scene> between RNase A and <scene name='~~Sandbox_Reserved_194~~/~~1rta_just_dt~~/~~2' target='0~~'>thymidylic acid tetramer (d(pT)4)</scene> ([[1rta]]) provides information about specificity of the binding pocket subunits, B0, B1, B2 and B3. Many interactions observed in this complex occur between amino acid residues and the nucleic acid backbone. Examples of these interactions include hydrogen bonding between <scene name='~~Sandbox_Reserved_194~~/~~1rta_structure_arg~~/~~5' target='0~~'> phosphate of T1 and Arg39</scene> as well as hydrogen bonding between the O5’ oxygen of the ribose of <scene name='~~Sandbox_Reserved_194~~/~~1rta_lys41~~/~~1' target='0~~'>T3 and Lys41</scene>. <ref>PMID:1429575</ref>	+	To determine the structural characteristics of RNA substrate binding to RNase A, X-ray crystallography was used to image inhibitory DNA tetramers bound to RNase A. DNA lacks the 2’OH essential to RNA cleavage, making the complex more conducive to crystallography. <scene name='44/449690/Cv/8'>The complex</scene> between RNase A and <scene name='44/449690/Cv/9'>thymidylic acid tetramer (d(pT)4)</scene> ([[1rta]]) provides information about specificity of the binding pocket subunits, B0, B1, B2 and B3. Many interactions observed in this complex occur between amino acid residues and the nucleic acid backbone. Examples of these interactions include hydrogen bonding between <scene name='44/449690/Cv/10'>phosphate of T1 and Arg39</scene> as well as hydrogen bonding between the O5’ oxygen of the ribose of <scene name='44/449690/Cv/12'>T3 and Lys41</scene>. <ref>PMID:1429575</ref>

	[[Image:1RCNnew.png\|thumb\|left\|200px\|ApTpApApG complexed with ribonuclease A]]		[[Image:1RCNnew.png\|thumb\|left\|200px\|ApTpApApG complexed with ribonuclease A]]

Alexander Berchansky at 10:43, 27 February 2019

Alexander Berchansky — Wed, 27 Feb 2019 10:43:42 GMT

←Older revision		Revision as of 10:43, 27 February 2019
Line 49:		Line 49:
	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 using three main catalytic residues (<scene name='44/449690/Cv/3'>His12, Lys41, and His119</scene> and <scene name='44/449690/Cv/4'>His12, Lys41, and His119 with substrate present</scene>). 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="Raines" />.		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 using three main catalytic residues (<scene name='44/449690/Cv/3'>His12, Lys41, and His119</scene> and <scene name='44/449690/Cv/4'>His12, Lys41, and His119 with substrate present</scene>). 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="Raines" />.

-	RNA hydrolysis begins when <scene name='44/449690/Cv/5'>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="Raines" />.	+	RNA hydrolysis begins when <scene name='44/449690/Cv/5'>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='44/449690/Cv/6'>Lys41</scene> and the main chain amide nitrogen of Phe120. <scene name='44/449690/Cv/7'>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="Raines" />.

	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="Raines" />.		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="Raines" />.

Alexander Berchansky at 10:38, 27 February 2019

Alexander Berchansky — Wed, 27 Feb 2019 10:38:00 GMT

←Older revision		Revision as of 10:38, 27 February 2019
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	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 using three main catalytic residues (<scene name='44/449690/Cv/3'>His12, Lys41, and His119</scene> and <scene name='44/449690/Cv/4'>His12, Lys41, and His119 with substrate present</scene>). 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="Raines" />.		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 using three main catalytic residues (<scene name='44/449690/Cv/3'>His12, Lys41, and His119</scene> and <scene name='44/449690/Cv/4'>His12, Lys41, and His119 with substrate present</scene>). 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="Raines" />.

-	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="Raines" />.	+	RNA hydrolysis begins when <scene name='44/449690/Cv/5'>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="Raines" />.

	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="Raines" />.		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="Raines" />.

Alexander Berchansky at 10:33, 27 February 2019

Alexander Berchansky — Wed, 27 Feb 2019 10:33:19 GMT

←Older revision		Revision as of 10:33, 27 February 2019
Line 47:		Line 47:
	==='''Acid Base Catalysis by RNase A'''===		==='''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 using three main catalytic residues (<scene name='44/449690/Cv/3'>His12, Lys41, and His119</scene> and <scene name='~~Sandbox_Reserved_194~~/~~His12_lys41_his119~~/1'>His12, Lys41, and His119 with substrate present</scene>). 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="Raines" />.	+	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 using three main catalytic residues (<scene name='44/449690/Cv/3'>His12, Lys41, and His119</scene> and <scene name='44/449690/Cv/4'>His12, Lys41, and His119 with substrate present</scene>). 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="Raines" />.

	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="Raines" />.		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="Raines" />.

Alexander Berchansky at 10:11, 27 February 2019

Alexander Berchansky — Wed, 27 Feb 2019 10:11:34 GMT

←Older revision		Revision as of 10:11, 27 February 2019
Line 47:		Line 47:
	==='''Acid Base Catalysis by RNase A'''===		==='''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 using three main catalytic residues (<scene name='~~Sandbox_Reserved_193~~/~~His12_lys41_his119~~/1'>His12, Lys41, and His119</scene> and <scene name='Sandbox_Reserved_194/His12_lys41_his119/1'>His12, Lys41, and His119 with substrate present</scene>). 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="Raines" />.	+	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 using three main catalytic residues (<scene name='44/449690/Cv/3'>His12, Lys41, and His119</scene> and <scene name='Sandbox_Reserved_194/His12_lys41_his119/1'>His12, Lys41, and His119 with substrate present</scene>). 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="Raines" />.

	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="Raines" />.		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="Raines" />.

Alexander Berchansky at 09:58, 27 February 2019

Alexander Berchansky — Wed, 27 Feb 2019 09:58:12 GMT

←Older revision		Revision as of 09:58, 27 February 2019
Line 36:		Line 36:
	RNase A uses acid/base catlysis to speed up RNA hydrolysis. This occurs in the <scene name='Sandbox_Reserved_193/Active_site_a/1'>active site</scene> which is found in the cleft of RNase A and is the location of the chemical change in bound substrates. Subsites lining the active site cleft are important to the binding of single stranded RNA. Large quantities of positively charged residues, such as <scene name='Sandbox_Reserved_193/Lys7_arg10_arg39_lys41_lys66/1'>Lys7, Arg10, Arg39, and Lys41, and Lys66</scene>, recognize the negative charge on the phosphate back bone of the <scene name='44/449690/Cv/1'>RNA strand</scene> <ref name="Wlodrawer" />.		RNase A uses acid/base catlysis to speed up RNA hydrolysis. This occurs in the <scene name='Sandbox_Reserved_193/Active_site_a/1'>active site</scene> which is found in the cleft of RNase A and is the location of the chemical change in bound substrates. Subsites lining the active site cleft are important to the binding of single stranded RNA. Large quantities of positively charged residues, such as <scene name='Sandbox_Reserved_193/Lys7_arg10_arg39_lys41_lys66/1'>Lys7, Arg10, Arg39, and Lys41, and Lys66</scene>, recognize the negative charge on the phosphate back bone of the <scene name='44/449690/Cv/1'>RNA strand</scene> <ref name="Wlodrawer" />.

-	The active site for RNase A, although fairly nonspecific, has some specificity for sites RNA hydrolysis. <scene name='~~Sandbox_Reserved_193~~/~~Thr45_a~~/1'>Threonine 45</scene>, located next to the active site, will hydrogen bond to pyrimidine bases, but sterically hinder the binding of a purine on the 5' strand of OH. Thr45 significantly decreases the rate of hydrolysis of polymeric purine strands, such as poly A, by a thousand fold, as compared to polymeric pyrimidine strands.<ref name = 'Wlodrawer'>PMID:3401445</ref>	+	The active site for RNase A, although fairly nonspecific, has some specificity for sites RNA hydrolysis. <scene name='44/449690/Cv/2'>Threonine 45</scene>, located next to the active site, will hydrogen bond to pyrimidine bases, but sterically hinder the binding of a purine on the 5' strand of OH. Thr45 significantly decreases the rate of hydrolysis of polymeric purine strands, such as poly A, by a thousand fold, as compared to polymeric pyrimidine strands.<ref name = 'Wlodrawer'>PMID:3401445</ref>

	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="Wlodrawer" />.		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="Wlodrawer" />.

Alexander Berchansky at 09:48, 27 February 2019

Alexander Berchansky — Wed, 27 Feb 2019 09:48:37 GMT

←Older revision		Revision as of 09:48, 27 February 2019
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	[[Image:MechIII.png\|400px\|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\|400px\|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'']]
	{{Clear}}		{{Clear}}
-	RNase A uses acid/base catlysis to speed up RNA hydrolysis. This occurs in the <scene name='Sandbox_Reserved_193/Active_site_a/1'>active site</scene> which is found in the cleft of RNase A and is the location of the chemical change in bound substrates. Subsites lining the active site cleft are important to the binding of single stranded RNA. Large quantities of positively charged residues, such as <scene name='Sandbox_Reserved_193/Lys7_arg10_arg39_lys41_lys66/1'>Lys7, Arg10, Arg39, and Lys41, and Lys66</scene>, recognize the negative charge on the phosphate back bone of the <scene name='~~Sandbox_Reserved_194~~/~~Positive_amino_acids_substrate~~/1'>RNA strand</scene> <ref name="Wlodrawer" />.	+	RNase A uses acid/base catlysis to speed up RNA hydrolysis. This occurs in the <scene name='Sandbox_Reserved_193/Active_site_a/1'>active site</scene> which is found in the cleft of RNase A and is the location of the chemical change in bound substrates. Subsites lining the active site cleft are important to the binding of single stranded RNA. Large quantities of positively charged residues, such as <scene name='Sandbox_Reserved_193/Lys7_arg10_arg39_lys41_lys66/1'>Lys7, Arg10, Arg39, and Lys41, and Lys66</scene>, recognize the negative charge on the phosphate back bone of the <scene name='44/449690/Cv/1'>RNA strand</scene> <ref name="Wlodrawer" />.

	The active site for RNase A, although fairly nonspecific, has some specificity for sites RNA hydrolysis. <scene name='Sandbox_Reserved_193/Thr45_a/1'>Threonine 45</scene>, located next to the active site, will hydrogen bond to pyrimidine bases, but sterically hinder the binding of a purine on the 5' strand of OH. Thr45 significantly decreases the rate of hydrolysis of polymeric purine strands, such as poly A, by a thousand fold, as compared to polymeric pyrimidine strands.<ref name = 'Wlodrawer'>PMID:3401445</ref>		The active site for RNase A, although fairly nonspecific, has some specificity for sites RNA hydrolysis. <scene name='Sandbox_Reserved_193/Thr45_a/1'>Threonine 45</scene>, located next to the active site, will hydrogen bond to pyrimidine bases, but sterically hinder the binding of a purine on the 5' strand of OH. Thr45 significantly decreases the rate of hydrolysis of polymeric purine strands, such as poly A, by a thousand fold, as compared to polymeric pyrimidine strands.<ref name = 'Wlodrawer'>PMID:3401445</ref>