Sandbox Reserved 192
| This Sandbox is Reserved from Feb 02, 2011, through Jul 31, 2011 for use by the Biochemistry II class at the Butler University at Indianapolis, IN USA taught by R. Jeremy Johnson. This reservation includes Sandbox Reserved 191 through Sandbox Reserved 200. |
To get started:
More help: Help:Editing |
Shown here is the active site of RNase A with catalytic residues highlighted.
|
IntroductionIntroduction
Ribonucleases [1] or RNA depolymerases are enzymes that catalyze RNA degradation. Ribonucleases are most commonly found in the pancreas because of their ability to digest large amounts of RNA excreted by the stomach. The pancreas in ruminants, such as cows, have especially high amounts of ribonucleases in order to process nutrients from cellulose based plants. One such ribonuclease, ribonuclease A or RNase A from cows, has been thoroughly studied due to its prevalence and 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. RNase A is in the shape of a kidney, with the active-site residues located within the cleft. residues and and aid in catalysis for the enzyme. Lys41 has been found to stabilize the negative charge in the transition state, while His12 likely acts as a base in catalysis and His119 likely acts as an acid in catalysis. Catalytic residues are shown together . The amino acid sequence determines the three-dimensional structure of RNase A based on side-chain interactions.
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 discusses the possibility of using the degradation capabilities of RNase A and a similar protein DNase I to treat tumors. Tumor propagation is associated with an imbalance in the level of nucleic acid degradation. This is shown by increased levels of nucleic acids and decreased levels of nuclease activity in the blood of patients. The high levels of nucleic acids are caused by the unregulated expression and the secretion of a specific tumor-derived miRNA and DNA. It was found that the combined treatment with the RNase A and the DNase produced the best results by slowing the growth rate of the tumor. But both the ribonucleases are toxic at high levels; thus, only low levels can be administered to prevent adverse effects.
Radicalization of RNase A: Tandem radical damage is a degenerative process where the radicalization of one molecule leads to a number of adverse biological effects. Methionine (Met) residues are transformed to alpha-aminobutyric acid (Aba) with a methanethiyl radical byproduct when bombarded with H*. When cysteine residues are radicalized, they are transformed to Alanine releasing sulfur radicals. This byproduct radical interact with the cis double bond in phospholipid fatty acids causing the conformational shift to the trans isomer. This switch from cis to trans causes many serious unfavorable biological diseases. Further studies into radical stresses on RNase A will help to better understand cellular degradation associated with aging and such degenerative pathologies.
|
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]