User:R. Jeremy Johnson/RNaseA: Difference between revisions

Ribonucleases [http://en.wikipedia.org/wiki/Ribonucleases] or RNA depolymerases are enzymes that catalyze RNA degradation. Ribonucleases are highly active in ruminants [http://en.wikipedia.org/wiki/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, bovine ribonuclease A or RNase A, is a model enzyme due to its ease of purification and simple structure.

RNase A has been used as a foundation enzyme for study due to its stability, small size, and because its three-dimensional structure is fully determined by its amino acid sequence, needing no molecular chaperones. 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. The previously mentioned Christian Anfinsen received the 1972 Nobel Prize in Chemistry for his paper "Principles that govern the folding of protein chains." Stanford Moore and William H. Stein received the 1972 Nobel Prize in Chemistry for their paper "The chemical structures of pancreatic ribonuclease and deoxyribonuclease." The 1984 Nobel Prize in Chemistry was awarded to Robert Bruce Merrifield for his paper "Solid-phase synthesis" using RNase A (Raines). 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 [http://en.wikipedia.org/wiki/X-ray_diffraction_analysis]. Disulfide bonds in RNase A were determined after developing a method using Fast Atom Bombardment Mass Spectrometry (FABMS) [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. The methods of NMR spectroscopy [http://en.wikipedia.org/wiki/NMR_spectroscopy] and Fourier transform infrared (FTIR) spectroscopy [http://en.wikipedia.org/wiki/Fourier_transform_infrared_spectroscopy]  were developed with RNase A in determining protein structure and protein folding pathways. These new methods, developed with RNase A, could be used for further research to determine the protein structure and protein folding pathways of other proteins (Raines).

{{STRUCTURE_7rsa |  PDB=7RSA |  SCENE= Sandbox_Reserved_193/Rnasei_a/1 }}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.

In organic chemistry acid/base catalysis is the addition of an acid or base to accelerate a chemical reaction. Ribonuclease A, (RNase A), also uses acid/base catatalysis to chemically change its substrates. Acidic or basic residues of the enzyme transfer protons to or from the reactant in order to stabilize the developing charges in the transition state. The transfer of protons usually creates better leaving groups, making the reaction more energetically favorable. Histidine is a very common amino acid residue involved in cataylsis, as histidine has a pKa value close to neutral, (p''K''a=6); therefore, histidine can both accept and donate protons at physiological pH.  

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.

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="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" />.  

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" />.

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" />.  

[[Image:RI.PNG|280px|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'']] 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 non-globular nature. RI is a 50 kD protein that is composed of 16 repeating subunits of alpha helices and beta sheets, giving it a noticable horseshoe like appearance. 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 RNases <ref name="Vicentini1996" />. 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 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" />

 

 

RNase variants have undergone duplication six times since amphibians and mammals diverged, giving rise to RNase A and other homologues. RNase A was believed to have become more specified within bovids[http://en.wikipedia.org/wiki/Bovid] 35 million years ago (Opitz et al. 1997). RNase A homologues have been found in frogs and humans by comparing the amino acid sequences of these particular enzymes with RNase A and seeing what residues were conserved. <scene name='Sandbox_Reserved_192/Conserved_residues/2'>Conservation of amino acid residues</scene>, 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.  However, these RNase A homologues differ in cytotoxicity and also have slight differences in sequences which may lead to different functions. One homologue, angiogenin, promotes neovascularization [http://en.wikipedia.org/wiki/Neovascularization]. Unusual homologues include other RNase homologues in the human body such as in urine and red blood cells. (Raines)

Another member in the ribonuclease family and structural homologue to bovine RNase A is frog onconase [http://en.wikipedia.org/wiki/Onconase] or ONC. ONC is found in oocytes [http://en.wikipedia.org/wiki/Oocytes] and early embryos of northern leopard frogs. The frog ribonuclease variant shows both cytostatic (cell growth suppression) and cytotoxic (prevents cell divisions) characteristics when it interacts with tumor cells. According to Gahl et al. (2008), no side effects have been determined for ONC. Leland et al. (2001) looked to determine the interactions that control the folding of ONC in order to develop effective mimics of ONC. In order to determine the interactions that controlled folding, the regeneration of RNase A was studied. Although RNase A and ONC were structurally very similar, there were significant differences in their folding pathways. While ONC forms a stable disulfide intermediate, RNase A does not. ONC was also found to be missing a disulfide bond that RNase A possesses. In the case of both enzymes, entropy is lost in the formation of the disulfide bonds, but folding may be driven by enthalpically favorable interactions of the side chains. Further experiments are being done to identify intramolecular interactions that account for the increased rate and formation of the structured intermediate in ONC (Gahl).