RNase A: Difference between revisions
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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 | 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. | ||
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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=' | 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> | ||
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