Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four-residue sequence recognized by factor Xa protease. The target was beta-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).
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PMID:Ribonuclease S-peptide as a carrier in fusion proteins. 845 73

For use in protein folding studies, ribonuclease A (RNase A), a 124-residue protein with the C-terminal sequence Phe-Asp-Ala-Ser-Val, has been labeled site-specifically by a carboxypeptidase Y (CPase Y)-catalyzed transpeptidation reaction which replaces the C-terminal residue(s) of RNase A with the extrinsic fluorescent probe 3-(2-naphthyl)-L-alanine amide (Nal-NH2). It was found that this CPase Y-catalyzed transpeptidation required the presence of the chemical denaturant urea (ca. 5 M) and that, under these conditions, effective transpeptidation occurred only in the pH range of ca. 5.5 to 6.5. The two major products from this labeling reaction were purified to homogeneity by ion-exchange and reverse-phase chromatography and characterized by tryptic mapping, amino acid analysis, and mass spectrometry. The major product, obtained in the reproducible, isolated molar yield of 20%, is the derivative in which only the single C-terminal residue, Val-124, is replaced directly by the probe Nal-NH2. Another isolated product, obtained in 11% yield, is the derivative in which two of the C-terminal residues, Ser-123 and Val-124, are replaced by one Nal-NH2. Both of these purified derivatives are enzymatically active (85 and 18%, respectively), and both exhibit spectral properties characteristic of the extrinsic probe including an observed fluorescence lifetime which is approximately monoexponential (tau = 42 ns) when the naphthyl chromophore is excited in the region of its absorption band.
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PMID:C-terminal labeling of ribonuclease A with an extrinsic fluorescent probe by carboxypeptidase Y-catalyzed transpeptidation in the presence of urea. 846 56

Escherichia coli RNase T, which is responsible for the 3' processing and end-turnover of tRNA and the maturation of 5 S RNA, is extremely sensitive to sulfhydryl reagents and to oxidation, suggesting a role for cysteine residues in its activity. Titration of homogeneous RNase T with 5,5'-dithiobis-(2-nitrobenzoic acid) revealed that the 4 cysteine residues present in each of the two protein subunits are in a reduced form and that 1 or 2 of them are important for activity. To identify these residue(s), each of the cysteines in RNase T was changed individually to either serine or alanine. The serine mutant at position 168 is greatly reduced in RNase T activity both in vivo and in vitro; likewise, the serine mutant at position 112 and the alanine mutants at positions 112 and 168 also display decreased RNase T activity. Mutations at the other cysteine positions show little or no change. Kinetic analyses of the mutant enzymes showed that the Km values of C168S and C168A are increased considerably, whereas their Vmax values are reduced only slightly compared to the wild type enzyme. The other mutant enzymes are little changed. Additional amino acid replacements at position 168 showed that the in vivo and in vitro activities of RNase T are in the order Cys approximately Val > Ala >> Ser >> Asn approximately Asp, which closely follows the relative hydrophobicity of these amino acid residues. However, the affinity for tRNA, determined by fluorescence quenching, is not altered in C168S, suggesting that Cys-168 is not directly involved in substrate binding. Interestingly, proteins altered at position 168 showed increased temperature sensitivity as the residue at that position became less hydrophobic. These data indicate that Cys-168 contributes a hydrophobic group that influences the structure and ultimately the catalytic activity of RNase T.
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PMID:The role of individual cysteine residues in the activity of Escherichia coli RNase T. 855 40

Barnase, the guanine specific ribonuclease of Bacillus amyloliquefaciens, was subjected to mutations in order to alter the electrostatic properties of the enzyme. Ser-85 was mutated into Glu with the goal to introduce an extra charge in the neighborhood of His-102. A double mutation (Ser-85-Glu and Asp-86-Asn) was introduced with the same purpose but without altering the global charge of the enzyme. A similar set of mutations was made using Asp at position 85. For all mutants the pI was determined using the technique of isoelectric focusing and calculated on the basis of the Tanford-Kirkwood theory. When Glu was used to replace Ser-85, the correlation between the experimental and the calculated values was perfect. However, in the Ser-85-Asp mutant, the experimental pI drop is bigger than the calculated one, and in the double mutant (Ser-85-Asp and Asp-86-Asn) the compensation is not achieved. The effect of the mutations on the pKa of His-102 can be determined from the pH dependence of the kcat/KM for the hydrolysis of dinucleotides, e.g., GpC. The effect can also be calculated using the the method of Honig. In this case the agreement is very good for the Glu-mutants and the single Asp-mutant, but less for the double Asp-mutant. The global stability of the Asp-mutants is, however, the same as the wild type, as shown by stability studies using urea denaturation. Molecular dynamics calculations, however, show that in the double Asp-mutant His-102 (H+) swings out of its pocket to make a hydrogen bridge with Gin-104 which should cause an additional pKa rise. The effect of the Glu-mutations was also tested on all the kinetic parameters for GpC and the cyclic intermediate G > p at pH 6.5, for RNA at pH 8.0, and for poly(A) at pH 6.2. The effect of the mutations is rather limited for the dinucleotide and the cyclic intermediate, but a strong increase of the KM is observed in the case of the single mutant (extra negative charge) with polymeric substrates. These results indicate that the extra negative charge has a strong destabilizing effect on the binding of the polymeric substrates in the ground state and the transition state complex. A comparison with the structure of bound tetranucleotides (Buckle, A.M. and Fersht, A.R., Biochemistry 33:1644-1653, 1994) shows that the extra negative charge points towards the P2 site.
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PMID:Experimental and theoretical study of electrostatic effects on the isoelectric pH and the pKa of the catalytic residue His-102 of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase). 877 84

The LH/CG receptor (LH/CG-R) belongs to the family of glycoprotein hormone G protein-coupled receptors. The extracellular domain of LH/CG-R is associated with high ligand-binding affinity and contains leucine-rich repeats (LRRs). With the goal of identifying essential amino acid residues involved in ligand binding, we replaced several conserved ionizable residues in the rat LH/CG-R with ones of opposite charge. The expression of these mutants was assessed by binding studies and Western blots after COS-7 cells were transiently transfected with wild type and mutant receptor cDNAs. The charge inversion of each of Lys40, Lys104, Asp118, Glu132, and Asp135 with Asp or Lys resulted in no detectable human CG binding in intact or solubilized cells; as control, a Lys40-->Arg replacement yielded a mutant with characteristics of the wild type receptor. Western analysis showed that the Lys40-->Arg mutant expressed at a level comparable to that of wild type receptor and, like wild type, exhibited a predominant immunoreactive mature form of LH/CG-R. Each of the five charge inversion mutants expressed at a lower level than wild type as assessed by immunoreactivity, and the levels of the Lys40-->Asp and Glu132-->Lys mutants were particularly low. The ratio of the mature to immature form of the receptor was high, i.e. like that of wild type, for the Glu132-->Lys and Asp135-->Lys replacements; the three other charge inversion mutants exhibited less mature than immature forms of the receptor. To aid in interpreting these results, we developed a model incorporating residues 27-235 of the extracellular domain of the rat LH/CG-R based on the crystal structure of the porcine ribonuclease inhibitor. Sequence homology and alignment revealed nine LRRs, with flanking cysteine clusters as found in a number of LRR proteins. Our model suggested that the Lys replacements of Glu132 and Asp135, i.e. those mutants that formed mature receptors, would disrupt the regional negative charge of the receptor. We propose that these residues are either directly involved in hormone binding or indirectly by disruption of the charge of an important binding surface.
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PMID:Determination of residues important in hormone binding to the extracellular domain of the luteinizing hormone/chorionic gonadotropin receptor by site-directed mutagenesis and modeling. 888 49

The human and murine pregnancy-specific glycoprotein (PSG) gene families encode a large number of closely related proteins which are abundantly expressed in the fetal trophoblast and secreted into the maternal circulation. Although the presence of a well conserved tripeptide sequence His or Arg-Gly-Asp or Glu or Lys (H/RGD/E/K) similar to the RGD motif found in extracellular matrix proteins hints towards a possible interaction with integrin-type receptors, the function of this group of proteins related to the carcinoembryonic antigen family is still unknown. It is also not clear whether the various members of the PSG family exert the same function. Here we describe the cloning of two splice variants of Cea4 (Cea4a, Cea4b), a murine PSG family member, which lacks the RGD-related consensus motif. Cea4a, like most of the other rodent PSG members, is composed of three immunoglobulin (Ig) variable-like domains (N1-N3) and and one Ig constant-like domain (A). In contrast, Cea4b lacks the N2 domain (N1N3A), demonstrating for the first time that PSG isoforms produced by alternative splicing also exist in mice. The mRNAs coding for Cea4a and Cea4b exhibit the same expression kinetics during placental development as found for two other murine PSGs, Cea2 and Cea3, which contain the RGD-like motif. Expression starts after day 12.5 of embryonic development (E12.5) and maximum steady-state levels are reached around E15.5-E17.5 as determined by RNase protection analyses. At E17.5, PSG transcripts can be detected exclusively in the spongiotrophoblast of the placenta. In addition, PCR analyses revealed that Cea2, Cea3, and Cea4 transcripts are also found in RNA from a pool of embryos (E12-E15) but are absent from a number of adult tissues tested (kidney, lung, testis, ovary, liver, brain, thymus, heart, spleen). These results indicate that the various PSG isoforms exert their function(s) at the same time during placental and embryonic development.
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PMID:Coordinate expression of splice variants of the murine pregnancy-specific glycoprotein (PSG) gene family during placental development. 897 44

RNase inhibitor (RI) binds with extraordinary affinity (Ki approximately 10(-13)-10(-16) M) to diverse proteins in the pancreatic RNase superfamily. In the present study, the structural basis for the recognition of two RI ligands, human angiogenin (Ang) and bovine RNase A, has been investigated by site-specific mutagenesis of human RI and Ang. The RI residues examined were those that appear to form strong contacts with RNase A in the crystal structure of the porcine RI x RNase A complex [Kobe, B. & Deisenhofer, J. (1995) Nature (London) 374, 183-186] that would not be replicated in the Ang complex. Ala substitutions of five of these residues (Glu-287, Lys-320, Glu-401, Cys-408, and Arg-457) were found to have little or no effect on binding of RNase A. In contrast, replacements of Tyr-434, Asp-435, and Tyr-437 and deletion of the C-terminal residue Ser-460 substantially weakened affinity for RNase A: the losses of binding energy associated with the mutations were 5.9, 3.6, 2.6, and 3.5 kcal/mol, respectively. Thus these four residues, which are neighbors in the tertiary structure, appear to constitute a "hot spot" for the RNase A interaction. However, only one of them, Asp-435, was equally important for binding of Ang; the Ki increases produced by mutations of the others were 20- to 93-fold smaller for Ang than for RNase A. Consequently, Tyr-434 plays a significant but lesser role in the Ang complex, whereas Tyr-437 and Ser-460 make only minor contributions. Ala mutations of four Ang residues (His-8, Gln-12, Asn-68, and Glu-108) that correspond to RI contacts on RNase A produced no major changes in affinity for RI. These findings indicate that RI uses largely different interactions to achieve its extremely tight binding of RNase A and Ang.
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PMID:Site-specific mutagenesis reveals differences in the structural bases for tight binding of RNase inhibitor to angiogenin and RNase A. 905 Aug 52

In order to investigate the specificity of peptide bond cleavage by acid proteinase A from Aspergillus niger var. macrosporus (Aspergillopepsin II), performic acid-oxidized bovine pancreatic ribonuclease A was digested by the enzyme at pH 1.8 or 5.5, and the resulting peptides were separated by HPLC and analyzed. Among the total 123 peptide bonds approximately thirty and thirteen bonds were cleaved at pH 1.8 for 2 h and at pH 5.5 for 20 h, respectively. Cleavages occurred fairly specifically at Tyr-X, Phe-X, His-X, Asn-X, Asp-X, Gln-X, and Glu-X bonds.
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PMID:The specificity of peptide bond cleavage of acid proteinase A from Aspergillus niger var. macrosporus toward oxidized ribonuclease A. 905 83

RNase LE from cultured tomato cells is a member of the RNase T2 family. It is, however, distinguishable from RNase Rh from Rhizopus niveus, a typical RNase of the RNase T2 family, by its CD spectrum in the 200-250 nm region. In order to reinvestigate the base specificity of RNase LE and to study the role of Asn44 in RNase LE, which is considered to correspond to the base recognition site Asp51 of RNase Rh, RNase LE, and its Asp mutant at the 44th position were expressed from yeast cells with the same expression system as RNase Rh [K. Ohgi, et al., J. Biochem., 109, 776-785 (1991)]. RNase LE with four extra amino acid residues at the 2nd amino acid residue of mature RNase LE and its Asp44 mutant were secreted from yeast cells to give a yield of 10 mg/liter and 0.5 mg/liter culture broth, respectively. The expressed RNase LE (RNase RNAP LE) had the same characteristics as native RNase LE in the CD spectrum and specific activity. This is the first example of the expression of plant RNase from microbes and in sufficient amount to perform further enzymological research. The base specificity of RNase LE was guanylic acid preferential and that of N44D was changed to a more adenylic acid preference as compared to that of RNase LE. These experiments showed that Asn44 of RNase LE is crucial for base recognition as the case of Asp51 in RNASE Rh, and also suggested that the base recognition mechanism of RNase LE is very similar to that of RNase Rh.
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PMID:The base specificities of tomato ribonuclease (RNase LE) and its Asp44 mutant enzyme expressed from yeast cells. 909 48

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.
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PMID:Human pancreatic ribonuclease--deletion of the carboxyl-terminal EDST extension enhances ribonuclease activity and thermostability. 915 80


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