Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oligosaccharide binding to chitosanase from Streptomyces sp. N174 was indirectly evaluated from thermal unfolding experiments of the protein. Thermal unfolding curves were obtained by fluorescence spectroscopy in the presence of D-glucosamine oligosaccharides ((GlcN)n, n = 3, 4, 5, and 6) using the inactive mutant chitosanase in which the catalytic residue, Glu22, is mutated to glutamine (E22Q), aspartic acid (E22D), or alanine (E22A). The midpoint temperature of the unfolding transition (Tm) of E22Q was found to be 44.4 degrees C at pH 7.0. However, the Tm increased upon the addition of (GlcN), by 1.3 degrees C (n = 3), 2.5 degrees C (n = 4), 5.2 degrees C (n = 5), or 7.6 degrees C (n = 6). No appreciable change in Tm was observed when (GlcNAc)6 was added to E22Q. The effect of (GlcN)n on the thermal stability was examined using the other protein, RNase T1, but the oligosaccharide did not affect Tm of the protein. Thus, we concluded that the stabilization effect of (GlcN)n on the chitosanase results from specific binding of the oligosaccharides to the substrate binding cleft. When E22D or E22A was used instead of E22Q, the increases in Tm induced by (GlcN)6 binding were 2.7 degrees C for E22D and 4.2 degrees C for E22A. In E22D or E22A, interaction with (GlcN)6 seems to be partly disrupted by a conformational distortion in the catalytic cleft.
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PMID:Substrate binding to the inactive mutants of Streptomyces sp. N174 chitosanase: indirect evaluation from the thermal unfolding experiments. 927 Dec 33

Our understanding of the factors stabilizing alpha-helical structure has been greatly enhanced by the study of model alpha-helical peptides. However, the relationship of these results to the folding of helices in intact proteins is not well characterized. Helix propensities measured in model peptides are not in good agreement with those from proteins. In order to address these questions, we have measured helix propensities in the alpha-helix of ribonuclease T1 and a helical peptide of identical sequence. We have previously demonstrated excellent agreement between peptide and protein for the nonpolar amino acids [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2833-2837]. Most other amino acids also show good agreement, although certain polar amino acids are exceptions. Helix propensities measured in the ribonuclease T1 peptide/protein are compared with those measured in other systems. Reasonable agreement is found between most systems; however, our propensities differ substantially from those measured in several model peptide systems. Alanine-based peptides overestimate the propensity differences by a factor of 2, and host/guest experiments underestimate them by a factor of 2-3.
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PMID:Helix propensities are identical in proteins and peptides. 928 83

Recently a new family of prolyl isomerases was discovered, which is unrelated with the cyclophilins or the FK-506 binding proteins. Parvulin, the smallest member of this new family, is a protein with only 92 residues, but parvulin-like domains occur in several large proteins that are apparently involved in protein folding or activation processes. We show here that, in addition to its activity in assays with proline-containing tetrapeptides, parvulin catalyzes the proline-limited folding of a variant of ribonuclease T1 with a kcat/Km value of 30,000 M-1 s-1. This value is much smaller than the kcat/Km value of 1.1x10(7) M-1 s-1 determined for the parvulin-catalyzed prolyl isomerization in the tetrapeptide succinyl-Ala-Leu-Pro-Phe-4-nitroanilide. Parvulin itself unfolds and refolds reversibly in a simple two-state reaction with a Gibbs free energy of stabilization of 28 kJ/mol at 10 degrees C. Most of the unfolded parvulin molecules refold in a slow reaction that involves prolyl isomerization and is catalyzed by cyclophilin, another prolyl isomerase. Moreover, parvulin accelerates its own refolding in an autocatalytic fashion, and the rate of refolding increases tenfold when the concentration of parvulin is increased from 0.5 to 3.0 microM. Apparently, small single-domain prolyl isomerases catalyze prolyl isomerization much better in short peptides than in protein folding reactions, presumably because the prolyl bonds are less accessible in refolding protein chains. It is possible that the additional domains of the large prolyl isomerases improve the affinity for protein substrates.
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PMID:Catalysis of protein folding by parvulin. 935 62

Trifluoroethanol (TFE) is often used to increase the helicity of peptides to make them usable as models of helices in proteins. We have measured helix propensities for all 20 amino acids in water and two concentrations of trifluoroethanol, 15 and 40% (v/v) using, as a model system, a peptide derived from the sequence of the alpha-helix of ribonuclease T1. There are three main conclusions from our studies. (1) TFE alters electrostatic interactions in the ribonuclease T1 helical peptide such that the dependence of the helical content on pH is lost in 40% TFE. (2) Helix propensities measured in 15% TFE correlate well with propensities measured in water, however, the correlation with propensities measured in 40% TFE is significantly worse. (3) Propensities measured in alanine-based peptides and the ribonuclease T1 peptide in TFE show very poor agreement, revealing that TFE greatly increases the effect of sequence context.
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PMID:Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T1. 952 Nov 15

Prolyl isomerases accelerate the cis <--> trans isomerization of prolyl peptide bonds during protein folding and probably also in folded proteins. We asked whether this catalytic function is in fact restricted to prolyl bonds or whether the isomerizations of 'normal' non-prolyl peptide bonds are catalyzed as well. By using the P39A variant of ribonuclease T1 as a substrate we find that the trans --> cis isomerization of the Tyr38-Ala39 bond in the refolding of this protein is not catalyzed by prolyl isomerases of the cyclophilin, FKBP and parvulin families. These enzymes are neither able to catalyze amide bond isomerizations in the proline-free model peptide Ala-Ala-Tyr-Ala-Ala.
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PMID:Prolyl isomerases do not catalyze isomerization of non-prolyl peptide bonds. 956 33

An antibody generated to an alpha-keto amide containing hapten 1 catalyzes the cis-trans isomerization of peptidyl-prolyl amide bonds in peptides and in the protein RNase T1. The antibody-catalyzed peptide isomerization reaction showed saturation kinetics for the cis-substrate, Suc-Ala-Ala-Pro-Phe-pNA, with a kcat/Km value of 883 s-1.M-1; the reaction was inhibited by the hapten analog 13 (Ki = 3. 0 +/- 0.4 microM). Refolding of denatured RNase T1 to its native conformation also was catalyzed by the antibody, with the antibody-catalyzed folding reaction inhibitable both by the hapten 1 and hapten analog 13. These results demonstrate that antibodies can catalyze conformational changes in protein structure, a transformation involved in many cellular processes.
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PMID:Antibody catalysis of peptidyl-prolyl cis-trans isomerization in the folding of RNase T1. 963 34

To elucidate the functional role of Arg82 and Arg86 in the enzyme activity of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant Arg86 Ala is 2.7 x 10(3) - 7.7 x 10(3) times less than that of the native enzyme. The decrease in activity is determined preferentially by the decrease in the molecular rate constant kcat with a relatively small change of enzyme-substrate affinity, characterized by Km. This is the expected result if Arg86 acts to lower the energy of a transition state of the reaction. The replacement of Arg82 by Ala causes a 5-19-fold activity decrease, depending on the substrate. We propose that this residue does not have a direct catalytic function in the molecular mechanism of the binase action and that the activity decrease of binase on the replacement of Arg82 by alanine is mediated by the effect of Arg82 on the pK of catalytic residues.
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PMID:Contribution of arginine-82 and arginine-86 to catalysis of RNases from Bacillus intermedius (binase). 964 74

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.
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PMID:Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1. 981 11

The mechanism by which barnase and binase are stabilized in their complexes with barstar and the role of the Cys-40 residue of barstar in that stabilization have been investigated by scanning microcalorimetry. Melting of ribonuclease complexes with barstar and its Cys-82-Ala mutant is described by two 2-state transitions. The lower-temperature one corresponds to barstar denaturation and the higher-temperature transition to ribonuclease melting. The barstar mutation Cys-40-Ala, which is within the principal barnase-binding region of barstar, simplifies the melting to a single 2-state transition. The presence of residue Cys-40 in barstar results in additional stabilization of ribonuclease in the complex.
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PMID:Key role of barstar Cys-40 residue in the mechanism of heat denaturation of bacterial ribonuclease complexes with barstar. 1009 94

The side-chain carboxyl of Asp 76 in ribonuclease T1 (RNase T1) is buried, charged, non-ion-paired, and forms three good intramolecular hydrogen bonds (2.63, 2.69, and 2.89 A) and a 2.66 A hydrogen bond to a buried, conserved water molecule. When Asp 76 was replaced by Asn, Ser, and Ala, the conformational stability of the protein decreased by 3.1, 3.2, and 3.7 kcal/mol, respectively. The stability was measured as a function of pH for wild-type RNase T1 and the D76N mutant and showed that the pH dependence below pH 3 was almost entirely due to Asp 76. The pK of Asp 76 is 0.5 in the native state and 3.7 in the denatured state. Thus, the hydrogen bonding of the carboxyl group of Asp 76 contributes more than half of the net stability of RNase T1 at pH 7. In addition, the charged carboxyl of Asp 76 stabilizes structure in the denatured states of RNase T1 that is not present in D76N, D76S, and D76A.
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PMID:Buried, charged, non-ion-paired aspartic acid 76 contributes favorably to the conformational stability of ribonuclease T1. 1052 13


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