Sandbox Reserved 192: Difference between revisions
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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 <scene name='Sandbox_Reserved_192/Disulfide_linkages/4'>disulfide linkages</scene>, 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. The structure of RNase A is often described as kidney shaped, with the active-site residues located within the cleft. <scene name='Sandbox_Reserved_192/Catalytic_residues/2'>His12, Lys41, and His119</scene> residues aid in catalysis. <scene name='Sandbox_Reserved_192/Lysine_41/3'>Lys41</scene> stabilizes the negative charge in the transition state, while <scene name='Sandbox_Reserved_192/His_12/3'>His12</scene> acts as a base and <scene name='Sandbox_Reserved_192/Histidine_119/2'>His119</scene> acts as an acid in catalysis. | 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 <scene name='Sandbox_Reserved_192/Disulfide_linkages/4'>disulfide linkages</scene>, 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. The structure of RNase A is often described as kidney shaped, with the active-site residues located within the cleft. <scene name='Sandbox_Reserved_192/Catalytic_residues/2'>His12, Lys41, and His119</scene> residues aid in catalysis. <scene name='Sandbox_Reserved_192/Lysine_41/3'>Lys41</scene> stabilizes the negative charge in the transition state, while <scene name='Sandbox_Reserved_192/His_12/3'>His12</scene> acts as a base and <scene name='Sandbox_Reserved_192/Histidine_119/2'>His119</scene> acts as an acid in catalysis. | ||
The amino acid sequence was found 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. 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. However, ensuing work showed some proteins require further assistance, such as molecular chaperones, to fold into their native structure. | The amino acid sequence was found 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. However, ensuing work showed some proteins require further assistance, such as molecular chaperones, to fold into their native structure. | ||
=='''History'''== | =='''History'''== | ||
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IntroductionIntroduction
Ribonucleases [1] or RNA depolymerases are enzymes that catalyze RNA degradation. Ribonucleases are highly active in ruminants, such as cows, to digest large amounts of RNA produced by microorganisms in the stomach. Ruminants also have high amounts of ribonucleases to process nutrients from cellulose. One such ribonuclease, ribonuclease A or RNase A from cows, is a model enzyme due to its ease of purification and simple structure.
StructureStructure
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 , which contribute to the stability of RNase A. Long four-stranded anti-parallel and three short make up the of RNase A. The structure of RNase A is often described as kidney shaped, with the active-site residues located within the cleft. residues aid in catalysis. stabilizes the negative charge in the transition state, while acts as a base and acts as an acid in catalysis. The amino acid sequence was found 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. However, ensuing work showed some proteins require further assistance, such as molecular chaperones, to fold into their native structure.
HistoryHistory
RNase A has been used as a foundation enzyme for study. The 1972 Nobel Prize in Chemistry was awarded to three researchers for their work with RNase A on the folding of chains in RNase A and the stability of RNase A. By mutating the residues of RNase A using site-directed mutagenesis [2], the effects of these mutations were more visibly analyzed with advances in analytical chemistry instrumentation and techniques. RNase A was the first enzyme and third protein for which its amino acid sequence was correctly determined and the third enzyme and fourth protein whose three-dimensional structure was determined by X-ray diffraction analysis. NMR spectroscopy [3] and Fourier transform infrared (FTIR) spectroscopy [4] also were used with RNase A to describe protein structure and protein folding.
Medical ImplicationsMedical Implications
Inhibition of Metastasis: A recent study published in 2010 revealed that tumor propagation is associated with an imbalance in nucleic acid metabolism. In the blood plasma of patients, there are increased levels of circulating nucleic acids and decreased nuclease activity. The abnormally high levels of circulating nucleic acids is associated with the increased expression and secretion of tumor-derived miRNA and DNA. With increased expression the tumor progresses and the patient has a bad prognosis.
RNase A and DNase I inhibit metastasis by catalyzing metastasis pathomorphosis which is apoptosis, necrosis and destruction of oncocytes (epithelial cells with large amounts of mitochondira). This capability retards the primary tumor growth by 30-40%. The tumor bearing mice received doses of RNase A, DNase I or a mixture of the two and the most significant effect observed was in the mice treated with both enzymes simultaneously. Thus the simultaneous administration of RNase A and DNase I lead to an anti-metastatic effect and results in an almost complete absence in the metastases of the tumor. Further observations suggest that RNase A and DNase I are toxic at high levels. So for effective treatment ultra low doses are required to stay below the level of toxicity.
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Further ResearchFurther Research
Replacement of Phe46: The phenylalanine-46 () residue located within the hydrophobic core of RNase A was experimentally replaced with other hydrophobic residues; leucine, valine and alanine. The x-ray crystallographic structures were determined in an attempt to conclude how the change would affect the conformational stability. It was concluded that the replacement of Phe46, which is key to the formation of the hydrophobic core, causes the destabilization of the RNase A by preventing the core from being tightly packed. But this has no effect on the substrates ability to bind. The core itself is very limited to adjusting to changes because of its limited mobility from the study di-sulfide bonds
Evolutionary SignificanceEvolutionary Significance
It has been determined that the amino acid sequence RNase A is quite similar to other proteins. , shown here for the homologues of RNase A, can either support or refute theories of protein structure and function. There have been over 40 different RNase homologues that have been sequenced. Conservation of amino acids Lys41 and His12 and His119 maintain the catalytic function within RNase A homologues. Unusual homologues include other RNase homologues in the human body such as in urine and red blood cells and those found from the eggs of bullfrogs. All RNase A homologues maintain the same function: to catalyze the cleavage of RNA.
Literary CitationsLiterary Citations
Ferreri, Carla; Chryssostomos Chatgillaloglu, Armida Torreggiani, Anna Marla Salzano, Giovanni Rensone, and Andrea Scaloni. “The Reductive Desulfurization of Met and Cys Residues in Bovine RNase A Is Assoicated with trans Lipid Formation in a Mimetic Model of Biological Membranes.” Journal of Proteom. 7 (2008): 2007-2015[5]
H. P. Avey; M. O. Boles; C. H. Carlisle; S. A. Evans; S. J. Morris; R. A. .Palmer; B. A. Woolhouse.”Structure of Ribonuclease.” Nature. 213 (1967) 557-562[6]
H. W. Wyckoff, Karl D. Hardman; N. M. Allewell; Tadash Inagam; L. N. Johnson. “The Structure of Ribonuclease-S at 3.5 A Resolution.” Department of Molecular Biophysics, Yale University. 242 (1967): 3984-3988 [7]
Kadonosono, Tetsuya; Eri Chatani, Rikimaru Hayashi, Hideaki Moriyama, and Tatzuo Ueki. “Minimization of Cavity Size Ensures Protein Stability and Folding: Structures of Phe-46-Replaced Bovine Pancreatic RNase A.” Biochemistry. 42 (2003): 10651-10658 [8]
Patutina, Olga; Nadezda Mironova, Elena Ryabchikova, Nelly Popova, Valery Nikolin, Vasily Kaledin, Valentin Valssov, Marina Zenkova. “ Inhibition of Metastasis Development by Daily Administration of Ultralow Doses of RNase A and DNase I” Biochimie. 93 (2011) 689-696 [9]
Raines, Ronald T. “Ribonuclease A.” Chemistry Review. Madison Wisconsin. 98 (1998): 1045-1065 [10]
Wlodawer, Alexander; L. Anders Svensson, Lennart Sjolin, Gary L. Gilliland. “Structure of Phosphate-Free Ribonuclease A Refined at 1.26 A.” American Chemical Society. 27 (1988) 2705-2717 [11]