Role of aspartic acid 121 in human pancreatic ribonuclease catalysis
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Keywords:
Bovine pancreatic ribonuclease
S-tag
Pancreatic ribonuclease
RNase PH
Alanine
Ribonuclease III
Aspartic acid
Angiogenin
Ribonuclease inhibitor (RI) is a cytoplasmic protein of ∼50 kDa that tightly binds and inhibits ribonucleases (RNases) from the pancreatic superfamily (). Diverse RNases with very limited sequence similarities, including RNase A, angiogenin, RNase-2 (also known as eosinophil-derived neurotoxin (EDN) or placental RNase), and RNase-4 are inhibited with K i values between 10−14 and 10−16 M. These affinities are among the highest reported for noncovalent binding of proteins. The binding occurs with 1∶1 stoichiometry. A subset of the pancreatic ribonuclease superfamily, including the amphibian ribonucleases such as frog-liver ribonuclease, sialic acid-binding lectin, and P-30 protein, are not inhibited by RI. The potent inhibitory activity of RI is believed to be utilized in RNA processing, angiogenesis, and protection of the cell from toxic ribonucleases.
Angiogenin
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Bovine pancreatic ribonuclease
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The primary structure of a non-secretory ribonuclease from bovine kidney (RNase K2) was determined. The sequence determined was VPKGLTKARWFEIQHIQPRLLQCNKAMSGV NNYTQHCKPENTFLHNVFQDVTAVCDMPNIICKNGRHNCHQSPKPVNLTQCNFIAGRYPDC RYHDDAQYKFFIVACDPPQKTDPPYHLVPVHLDKYF. The sequence homology with human non-secretory RNase, bovine pancreatic RNase, and human secretory RNase are 46, 34.6, and 32.3%, respectively. The bovine kidney RNase has two inserted sequences, a tripeptide at the N-terminus and a heptapeptide between the 113th and 114th position of bovine pancreatic RNase; on the other hand, it is deleted of the hexapeptide consisting of the 17th to the 22nd amino acid residue of RNase A. The amino acid residues assumed to be the constituents of the bovine pancreatic RNase active site are all conserved except F120 (L in RNase K2).
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ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMutagenesis of residues flanking Lys-40 enhances the enzymic activity and reduces the angiogenic potency of angiogeninJ. Wade Harper, Edward A. Fox, Robert Shapiro, and Bert L. ValleeCite this: Biochemistry 1990, 29, 31, 7297–7302Publication Date (Print):August 7, 1990Publication History Published online1 May 2002Published inissue 7 August 1990https://pubs.acs.org/doi/10.1021/bi00483a020https://doi.org/10.1021/bi00483a020research-articleACS PublicationsRequest reuse permissionsArticle Views62Altmetric-Citations17LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Angiogenin
Bovine pancreatic ribonuclease
Pancreatic ribonuclease
S-tag
Cite
Citations (26)
Bovine pancreatic ribonuclease
S-tag
Pancreatic ribonuclease
RNase PH
Alanine
Ribonuclease III
Aspartic acid
Angiogenin
Cite
Citations (4)
A ribonuclease (RNase) that cleaves specifically on the 3′ side of uridine [Shapiro, R., Fett, J. W., Strydom, D. J. & Vallee, B. L. (1986a) Biochemistry 25 , 7255–7264] was purified from human plasma and its amino acid sequence was determined. This protein is a 119‐residue single‐chain polypeptide cross‐linked by four disulfide bonds and has an amino‐terminal pyroglutaminyl residue. No post‐translational modifications were observed during extensive sequence studies on peptide fragments, except for the amino‐terminal pyroglutamic acid and a possible deamidation of Asn66. The protein is homologous to the pancreatic ribonucleases and angiogenin, but differs substantially from both of these proteins; the protein sequence has 43% identity with human pancreatic ribonuclease and 39% identity with human angiogenin, as compared to 35% identity between human angiogenin and pancreatic ribonuclease. It is referred to as RNase 4, based on the nomenclature currently used for the genes of pancreatic RNase (RNase 1) and the eosinophil‐derived RNases (RNase 2 and RNase 3). Virtually all of the RNase active‐site components, including the catalytic residues His12, His119 and Lys41, are preserved. However, some invariant residues of RNase 1 are replaced, e.g. Lys7 by arginine, Asp14 by histidine, and Pro42 by arginine. RNase 4 contains a unique two‐residue deletion at the position corresponding to amino acids 77 and 78 of pancreatic RNase, and its carboxy‐terminal sequence is truncated at position 122. The deletion in angiogenin at position 21 is also found in RNase 4. RNase 4 is very similar to two RNases isolated from bovine and porcine liver, and together they form a new family in the RNase superfamily. The degree of inter‐species similarity (90%) is much greater than within the pancreatic RNase and angiogenin families, which suggests that this ribonuclease could possess a physiologically important function other than general RNA catabolism.
Angiogenin
Pancreatic ribonuclease
RNase PH
Ribonuclease III
S-tag
RNase MRP
Bovine pancreatic ribonuclease
RNase H
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A comparison of the sequences of three homologous ribonucleases (RNase A, angiogenin and bovine seminal RNase) identifies three surface loops that are highly variable between the three proteins. Two hypotheses were contrasted: (i) that this variation might be responsible for the different catalytic activities of the three proteins; and (ii) that this variation is simply an example of surface loops undergoing rapid neutral divergence in sequence. Three hybrids of angiogenin and bovine pancreatic ribonuclease (RNase) A were prepared where regions in these loops taken from angiogenin were inserted into RNase A. Two of the three hybrids had unremarkable catalytic properties. However, the RNase A mutant containing residues 63–74 of angiogenin had greatly diminished catalytic activity against uridylyl-(3′ – 5′)-adenosine (UpA), and slightly increased catalytic activity as an inhibitor of translation in vitro. Both catalytic behaviors are characteristic of angiogenin. This is one of the first examples of an engineered external loop in a protein. Further, these results are complementary to those recently obtained from the complementary experiment, where residues 59–70 of RNase were inserted into angiogenin [Harper and Vallee (1989) Biochemistry, 28, 1875–1884]. Thus, the external loop in residues 63–74 of RNase A appears to behave, at least in part, as an interchangeable ‘module’ that influences substrate specificity in an enzyme in a way that is isolated from the influences of other regions in the protein.
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Bovine pancreatic ribonuclease
Pancreatic ribonuclease
S-tag
RNase PH
RNase H
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Mammalian ribonucleases constitute one of the fastest evolving protein families in nature. The addition of a four‐residue carboxyl‐terminal tail: Glu‐Asp‐Ser‐Thr (EDST) in human pancreatic ribonuclease (HPR) in comparison with bovine pancreatic RNase (RNase A) could have adaptive significance in humans. We have cloned and expressed human pancreatic ribonuclease in Escherichia coli to probe the influence of the four‐residue extension and neighboring C‐terminal residues on the biochemical properties of the enzyme. Removal of the C‐terminal extension from HPR yielded an enzyme, HPR‐(1–124)‐peptide, with enhanced ability to cleave poly(C). HPR‐(1–124)‐peptide also exhibited a steep increase in thermal stability mimicking that known for RNase A. Wild‐type HPR had significantly low thermal stability compared to RNase A. The study identifies the C‐terminal boundary in the human pancreatic ribonuclease required for efficient catalysis.
Bovine pancreatic ribonuclease
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Cleave
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Ribonuclease III
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RNase PH
Pancreatic ribonuclease
RNase H
Bovine pancreatic ribonuclease
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RNase MRP
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A variant of bovine pancreatic ribonuclease A has been prepared with seven amino acid substitutions (Q55K, N62K, A64T, Y76K, S80R, E111G, N113K). These substitutions recreate in RNase A the basic surface found in bovine seminal RNase, a homologue of pancreatic RNase that diverged some 35 million years ago. Substitution of a portion of this basic surface (positions 55, 62, 64, 111 and 113) enhances the immunosuppressive activity of the RNase variant, activity found in native seminal RNase, while substitution of another portion (positions 76 and 80) attenuates the activity. Further, introduction of Gly at position 111 has been shown to increase the catalytic activity of RNase against double‐stranded RNA. The variant and the wild‐type (recombinant) protein were crystallized and their structures determined to a resolution of 2.0 Å. Each of the mutated amino acids is seen in the electron density map. The main change observed in the mutant structure compared with the wild‐type is the region encompassing residues 16–22, where the structure is more disordered. This loop is the region where the polypeptide chain of RNase A is cleaved by subtilisin to form RNase S, and undergoes conformational change to allow residues 1–20 of the RNase to swap between subunits in the covalent seminal RNase dimer.
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Bovine pancreatic ribonuclease
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Pancreatic ribonuclease
RNase MRP
Ribonuclease III
RNase H
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Angiogenin (ANG) promotes the formation of blood vessels in animals. This hormone is a small, monomeric protein that is homologous to bovine pancreatic ribonuclease A (RNase). ANG is a poor ribonuclease but its ribonucleolytic activity is essential for its angiogenic activity. RNase is not angiogenic. A hybrid protein was produced in which 13 residues of a divergent surface loop of ANG were substituted for the analogous 15 residues of RNase. The value of kcat/Km for the cleavage of uridylyl(3′→5′)adenosine by this hybrid protein was 20-fold less than that of RNase but 105-fold greater than that of ANG. The thermal stability of the hybrid protein was also less than that of RNase. Nevertheless, the RNase/ANG hybrid protein promotes angiogenesis in mice at least as extensively as does authentic ANG. Thus we present a protein endowed with a noncognate biological activity simply by replacing a single element of secondary structure. In addition, a 13-residue peptide corresponding to the surface loop of ANG inhibits endogenous angiogenesis in mice. These results support a model in which both a surface loop and a catalytic site are necessary for the promotion of blood vessel formation by ANG or RNase. The dissection of structure/function elements in ANG reveals a unique opportunity to develop new molecules that modulate neovascularization. Angiogenin (ANG) promotes the formation of blood vessels in animals. This hormone is a small, monomeric protein that is homologous to bovine pancreatic ribonuclease A (RNase). ANG is a poor ribonuclease but its ribonucleolytic activity is essential for its angiogenic activity. RNase is not angiogenic. A hybrid protein was produced in which 13 residues of a divergent surface loop of ANG were substituted for the analogous 15 residues of RNase. The value of kcat/Km for the cleavage of uridylyl(3′→5′)adenosine by this hybrid protein was 20-fold less than that of RNase but 105-fold greater than that of ANG. The thermal stability of the hybrid protein was also less than that of RNase. Nevertheless, the RNase/ANG hybrid protein promotes angiogenesis in mice at least as extensively as does authentic ANG. Thus we present a protein endowed with a noncognate biological activity simply by replacing a single element of secondary structure. In addition, a 13-residue peptide corresponding to the surface loop of ANG inhibits endogenous angiogenesis in mice. These results support a model in which both a surface loop and a catalytic site are necessary for the promotion of blood vessel formation by ANG or RNase. The dissection of structure/function elements in ANG reveals a unique opportunity to develop new molecules that modulate neovascularization.
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