Ribonuclease A
Created by Tin Nguyen
Ribonuclease A-Uridine Vanadate Complex (1RUV) from Bos taurus is a complex of bovine ribonuclease A and uridine vanadate. Ribonuclease A is a small protein – more specifically, an endonuclease – consisting of a single chain of 124 amino acids that catalyzes the hydrolysis of the P-O5’ linkages in single-stranded RNA, which covalently link ribonucleotides to pyrimidine bases, such as uracil (1). UV-RNase A is specified in the pancreatic tissues and secreted by the pancreas to degrade ingested RNA, which is essential for cellular metabolism (2). Uridine vanadate (U-V) is a competitive inhibitor of Ribonuclease A; it is a stable isosteric analog of the pentacoordinate transition state for the cleavage of the phosphate-ester linkages by Ribonuclease A (3). The molecular weight of Ribonuclease A is 13,690.29 Da and its isoelectric point (pI) is 8.64 (5).
The structure of Ribonuclease A correlates to its function. The two ligand components for UV-RNase A are Tertiary-Butyl Alcohol and Uridine-2’-3’-Vanadate. Uridine vanadate-ribonuclease A complex exists physiologically as a monomer with four disulfide bonds. The four disulfide bonds are important to the stability of the native Ribonuclease A (2). The first two disulfide bonds (Cys-26 – Cys-84, and Cys-58 – Cys-110) are responsible for the conformational folding of the protein (6). Ribonucelase A contains 19 of the 20 natural amino acids, lacking tryptophan, and is a polar molecule. The overall shape of the protein has the active-site residues lying in the cleft. The enzyme has six substrate binding subsites. However, the uridine vanadate binding site of the Ribonuclease A is divided into three regions: P1, R1, and B1 subsite, which corresponds to the uridine base, the ribose ring, and the TBP vanadate, respectively (1).
Three of the subsites interact with the bases of a bound substrate; they include: B1, B2, and B3. The B1 pyrimidine-binding subsite is formed by the following residues: His-12, Val-43, Asn-44, Asn-45, Arg-83, and Phe-120. The B1 subsite only binds to pyrimidine bases and has a 30-fold kinetic preference for cytosine over uracil substrates. On the other hand, the enymic subsites B2 and B3 bind to all bases; B2, however, have a preference for adenine and B3 has a preference for purine (2). The other three enzymic subsites include: P0, P1, and P2, which interact with the phosphoryl groups of a bound substrate (2). The P1 subsite of UV-RNase A contains Gln-11, His-12, His-119, Lys-41, and Phe-120 (1). Ribonuclease A catalyses the cleavage of the P-O5’ bond of a phosphoryl group bound in the P1 subsite, known as the active site. In this P1 subsite, His-12, His-119, and Lys-41 are the main residues that participate in the catalytic processes (7). His119, Gln11, and Lys41 are three residues that interact directly with the uridine vanadate. Ribonuclease A contains four histidine residues: His-12, His-48, His-105, and His-119 (1). The R1 subsite interacts with the ribose and includes the following amino acids: His12, Lys41, Val43, His119, and Phe-120 (1). In the structure of UV-RNase, the side chain of Phe-120 has van der waals contact with a pyrimidine base bound in the B1 enzymic subset. The side chain of Ser-123 forms a hydrogen bond to a uracil bound in the B1 subsite, which improves the rate of cleavage after uridine residues (2).
The secondary structure of the enzyme contains a long four-stranded antiparallel β-sheet and three short α-helixes, with an extensive network of hydrogen bonds, which help stabilize the molecule. According to DSSP, UV-RNase A secondary structure is 20% helical (4 helices, 26 residues) and 35% β-sheet (10 strands, 44 residues) (9). The three helical regions of Ribonuclease A can consist of residues 3-13, 24-34, and 50-60. A larger part of the enzyme consists of β-sheets, where the backbone of the ribonuclease molecule consists of a pair of antiparallel β strand, which consists of residues 71-92 and 94-110. The strands are partially twisted, with residues 77 and 104 located at the apices. Residues 76-78 do not participate in the hydrogen bonding, however (10).
Ribonuclease A is cross-linked by four disulfide bonds, which are critical in maintaining the stability of the native enzyme (2). Each disulfide bond involves all eight of its cysteine residues. The two disulfide bonds (between Cys-26 and Cys-84, as well as between Cys-58 and Cys-110) contribute more to thermal stability than the disulfide bonds between Cys-40 and Cys-95 (2). The importance of the disulfide bonds for Ribonuclease A provides use probes for elaborating pathways for protein folding (2).
The three-dimensional structure of the enzyme is completely encoded by its amino acid sequence (2). One functionally important residue is Lys-41. Lys-41 is in the active site of the enzyme and it interacts directly with the vanadate O atoms in the Ribonuclease A-Uridine Vanadate Complex, which become less mobile upon U-V binding (1). Lys-41 has a catalytic role in stabilizing the excess negative charge on the nonbridging phosphoryl oxygens during RNA cleavage. Stabilization occurs by involving a short, strong hydrogen bond that involves a partial transfer of a proton from Lys-41 (11). The stability of the enzyme can be attributed to Lys-41 and its positive charge and ability to hydrogen bond.
Gln-11, found in the P1 subsite of Ribonuclease A, also has an important role in the catalytic process. Interestingly, Gln-11 is conserved in all of the 41 known pancreatic ribonuclease sequences (3). X-ray diffraction analysis suggests that Gln-11 forms a hydrogen bond to substrate, as well as substrate analogs, phosphate ions, or sulfate ions bound in the active site of the enzyme. Unlike Lys-41, Gln-11 does not stabilize the rate-limiting transition state during the catalytic process by Ribonuclease A. Gln-11 does, however, increase the free energy of the enzyme-substrate complex. The hydrogen bond between Gln-11 side chain and a phosphoryl oxygen indeed enhances catalysis by preventing the substrate from biding in a non-productive mode through reorientation (3). The active site is more likely to bind to an RNA molecule with its phosphyrl group in a wrong conformation when Gln-11 is absent (2). Therefore, the hydrogen bond between the side-chain of Gln-11 and the phosphoryl oxygen enhances the catalysis by orientating the substrate.
In addition to Lys-41 and Gln-11, histidines – specifically, His-12 and His-119 – are also important residues in the catalytic processes of Ribonuclease A. Ribonuclease contains four histidine residues: His-12, His-48, His-105, and His-119. His-12 and His-119 lie within the active site of the enzyme and function as acid/base donors in the catalysis mechanism (4). His-119 protects the leaving group during RNA cleaveage, while His-12 acts as a base during the catalytic mechanisms (2). In order to understand the importance of His-12 and His-119, it is important to look further into the enzymatic mechanism of Ribonuclease A. The catalytic process of Ribonuclease A occurs via a two-step acid/base mechanism, involving the intramolecular transphosphorylation forming a cyclic phosphate intermediate and displacement of the O5’ nucleotide product, followed by the hydrolyzation of a cyclic phosphate to a 3’-monophosphate nucleotide as final product (12). Uridine vanadate is an analog of the pentacoordinate transition state in the enzyme, and is known to lock the enzyme in specific position (4). His-12 and His-119 are important players in this catalytic process and act as the general base and acid, respectively, in the transphosphorylation step, and reversing their roles in the latter step (12). In the transphosphorylation step, His-12 acts as a base and removes a proton from the 2’ oxygen of the ribose; this results in O2’ oxygen attacking the phosphorous.
His-119, however, acts as an acid and donates a proton the O5’ of the phosphodiester. The second step of mechanism, His-119, reversely, acts as a base and accepts a proton from water, which attacks the 2’,3’-cyclic phosphate. This forms the pentacoordinate phosphorous. His-12 then acts as an acid to donate a proton to the O2’ atom and, therefore, forms a 4’ nucleotide (4). The imidazole ring of His-12 has a low pK value, suggesting that it must be deprotonated, while the imidazole ring of His-119 has a high pK value, suggesting it must be protonated for catalysis. In addition, His-12 and His-119 are suggested to be involved in the complexing with uridine vanadate (4). Furthermore, Lys-41 assists the deprotonation of 02’ by His-12, thus enhancing the deprotonation and inhibiting the protonation during hydrolysis (13). By acting as a base and abstracting a proton from the 2’-oxygen of the substrate molecule, the side chain of Lys-41 also enhance catalysis by stabilizing the transition state (2).
Another residue worth mentioning is Asp-121, which forms a hydrogen bond with His-119 in the native enzyme (14). The interaction between His-119 and Asp-121 defines a catalytic dyad. The histidine residue (His-119) mediates general acid-base catalysis with hydrogen bonds to an aspartate residue (Asp-121). Researchers studied the role of Asp121 in the catalytic dyad of Ribonuclease A and found that when Asp-121 was replaced with either an asparagine or alanine residue, there was a loss of conformational stability at pH 6.0 of ??G(0) = 2.0 k cal/mol, from a total of ?G(0) = 9.0 kcal/mol) (14). The loss is similar to the transition-state binding during the catalytic steps of RNA cleavage. Therefore, one major function of the His---Asp catalytic dyad is to improve the stability of the enzyme (2).
By comparing Ribonuclease A-Uridine Vandate Complex (pdb ID = 1RUV) to its unligated form, Ribonuclease A (pdb ID = 7RSA), additional insight into the enzyme’s structural variation induced by the uridine vanadate binding can be acknowledged. The two structures are very similar, aside from the displacement of the water molecules in the active site (1). The comparison between the two ligated and unligated molecule shows that the residues near the active site becomes less mobile after uridine vanadate binding, exhibiting reduced B factors. As previously mentioned, the three amino acids that are greatly directed with the U-V are: Gln-11, Lys-41, and His-119 (1). The presence of two conformations in Ribonuclease A (pdb ID = 7RSA) may be a result form not having a substrate or ligand present in the active site (1). The residues that exist with multiple conformations in the unligated molecule, but with only one conformation when bound to uridine vandate are as follow: Gln11, Asn-34, Val-43, Lys-61, Asn-67, Arg-85, Lys-91, Lys-98, and Lys-104 (1). Gln11 and Val43 interact indirectly with the uridine vanadate in the active site, while Arg-84 has van der waals interactions with Val43, which becomes conformationally constrained as uridine vanadate binds (1). Lys-91, Lys-98, and Lys-104 have increased B factors, thus showing high mobility making it hard to model additional conformation of the side chains (1).
Ribonuclease 7 (pdb ID= 2HKY) in Homo sapiens has a query coverage of 95% to ribonuclease A. The results of DALI (Z=13.9, rmsd=2.1) and protein Blast (E=1.22e-52) searches show that Ribonuclease 7 has secondary and tertiary structural similarities to Ribonuclease A. The structural differences lie in the length of the amino acid residues. Both proteins are small, but Ribonuclease 7 is 129 amino acids long compared to the 124 amino acid length of Ribonuclease A found in Bos taurus. Ribonuclease 7 has no ligands. Its molecular weight is 14685.1 Da and its theoretical isoelectric point (pI) is 9.80, compared to a pI of 8.54 for Ribonuclease A (5). Ribonucleaes A (pdb ID=1RRA) found in Rattus norvegicus has an approximate 80% sequence similarity to ribonuclease A (pdb ID=1RUV) in Bos taurus. The results of DALi (Z=21.7, rmsd=1.0) and protein Blast (E=5.71e-73) also suggest that Ribonuclease A in Rattus norvegicus has secondary and tertiary structural similarities to Ribonuclease A found in Bos taurus. The ligand found in Ribonuclease of comparison is a phosphate ion. The length of the comparison protein is the same as the query protein: 124 amino acid residues. Its molecular weight is 13,735.42 Da and has an isoelectric point (pI) of 8.65, compared to a molecular weight of 13,690.29 Da and a pI of 8.64 (5).
Ribonuclease A is a great molecular enzyme that degrades RNA; however, it is also toxic to cells. Cells protect themselves from the catalytic actions of ribonuclease with inhibitors, such as the ribonuclease inhibitors (2). The ribonuclease inhibitor is a 50 kDa protein that is less than 0.01% of the protein in the cytosol of mammalian cells (2). The ribonuclease inhibitor protects the RNA against invasion of secreted ribonuclease from the pancreas. This ability of ribonuclease inhibitor to protect RNA is an important aspect of research regarding Ribonuclease A. In addition, small-molecule inhibitors of Ribonuclease A include uridine 2’3’-cyclic vanadate, which is a potent inhibitor of Ribonuclease A. The Uridine-vanadate complex has a trigonal bipyramidal geometry when bound to the active site of the enzyme (2).