Kinetic studies of guanine recognition and a phosphate group subsite on ribonuclease T 1 using substitution mutants at Glu46 and Lys41 (original) (raw)
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Ribonuclease PH interacts with an acidic ribonuclease E site through a basic 80-amino acid domain
FEMS Microbiology Letters, 2014
In this work, we characterize the domains for the in vivo interaction between ribonuclease E (RNase E) and ribonuclease PH (RNase PH). We initially explored the interaction using pull-down assays with full wild-type proteins expressed from a chromosomal monocopy gene. Once the interaction was confirmed, we narrowed down the sites of interaction in each enzyme to an acidic 16-amino acid region in the carboxy-terminal domain of RNase E and a basic 80-amino acid region in RNase PH including an a3 helix. Our results suggest two novel functional domains of interaction between ribonucleases.
Protein Science, 2006
A general acid-base catalytic mechanism is responsible for the cleavage of the phosphodiester bonds of the RNA by ribonuclease A (RNase A). The main active site is formed by the amino acid residues His12, His119, and Lys41, and the process follows an endonucleolytic pattern that depends on the existence of a noncatalytic phosphate-binding subsite adjacent, on the 39-side, to the active site; in this region the phosphate group of the substrate establishes electrostatic interactions through the side chains of Lys7 and Arg10. We have obtained, by means of site-directed mutagenesis, RNase A variants with His residues both at positions 7 and 10. These mutations have been introduced with the aim of transforming a noncatalytic binding subsite into a putative new catalytic active site. The RNase activity of these variants was determined by the zymogram technique and steady-state kinetic parameters were obtained by spectrophotometric methods. The variants showed a catalytic efficiency in the same order of magnitude as the wild-type enzyme. However, we have demonstrated in these variants important effects on the substrate's cleavage pattern. The quadruple mutant K7H/R10H/H12K/H119Q shows a clear increase of the exonucleolytic activity; in this case the original native active site has been suppressed, and, as consequence, its activity can only be associated to the new active site. In addition, the mutant K7H/R10H, with two putative active sites, also shows an increase in the exonucleolytic preference with respect to the wild type, a fact that may be correlated with the contribution of the new active site.
FEBS Letters, 1994
Structures of a semisynthetic RNase have been obtained to a resolution of 2.0 8, at pH values of 5.2, 6.5, 7.5, and 8.8, respectively. The principle structural transformation occurring over this pH range is the conversion of the side chain of active site residue His-l 19 from one conformation k, = -43" to -57") at low pH to another k, = + 159" to + 168") at higher pH values. On the basis of this observation, a model is proposed that reconciles the disparate pK values for His-l 19 in the enzyme-substrate complex that have been deduced from kinetic studies and from proton NMR measurements in the presence of pseudosubstrates.
Ribonuclease Specificity: Combined Action of the Major Components of Crystalline Ribonuclease
Journal of Biological Chemistry, 1957
The actions of the naturally occurring ribonuclease (RNase) fractions, ribonuclease A (RNase A) and ribonuclease B (RNase B) , in certain defined proportions, determine the synthetic and hydrolytic activities of crystalline RNase. When the proportions of RNase A to RNase B are altered by artificial admixture, the activities of the whole enzyme (RNase) are different from those which are observed with the isolated enzyme. In previous investigations (1, 2), we showed that RNase B liberated 9.8 to 13.8 per cent of guanylic acid, and that crystalline ribonuclease, which contained both fractions A and B, liberated only 0.9 to 1.2 per cent. Furthermore, some synthetic activities of crystalline RNase observed by Heppel, Whitfeld, and Markham (3, 4) were demonstrated by us (2) to occur with RNase B but not RNase A. The complex nature of the action of crystalline RNase appears to depend upon the proportion of the two fractions, A and B, acting together. The action of RNase A and RNase B each alone and combined in various proportions has been investigated by paper chromatography and by electrotitration, and the results obtained are reported in the present paper. EXPERIMENTAL Materials Crystalline ribonucleases were prepared in this laboratory from calf pancreas (5) or were obtained from Armour and Company, the Worthington Biochemical Corporation, and the Nutritional Biochemicals Corporation. Yeast ribonucZeic acid (RNA) was prepared as reported previously (6). Methods Crystalline RNase "Armour," "Worthington," "Nutritional Biochemicals," and "Laboratory"' were each chromatographed on a column of car-1 RNase A and RNase B used in these investigations were obtained mainly from Armour crystalline RNase. The action of the crystalline enzymes "Armour," "Worthington, " "Nutritional Biochemicals," and "Laboratory" reported in Table I is taken from Hakim (2) for comparative purposes. 459 This is an Open Access article under the CC BY license.
Enzymic heterogeneity of crystalline ribonuclease
Archives of Biochemistry and Biophysics, 1957
Enzymic heterogeneity of crystalline ribonuclease has not been described to date, in spite of intensive investigations of the specificity of the enzyme as determined by the hydrolytic products which result from the action of ribonuclease on ribonucleic acid. Reports on the synthetic activities of ribonuclease by Heppel et al. (1, 2)) did not take into consideration the fact that crystalline ribonuclease has been separated into two fractions by partition (3)) ion-exchange resin chromatography (4)) and electrophoresis (5). In the present paper we demonstrate secondary reactions which differentiate the two major fractions of ribonuclease. MATERIALS AND METHODS Materials Four samples of ribonuclease were obtained either by preparation in crystalline form from calf pancreas (6) or by purchase from Armour and Co., Worthington Chemical Co., or Nutritional Biochemicals Corp. The term "crystalline ribonuclease", as used in this report, refers to each of the four different samples, prior to any fractionation. Spleen phosphodiesterase was obtained according to the method described by Heppel, Whitfeld, and Markham (2). Yeast ribonucleic acid was prepared as described by Hakim (7). Uridine d',S'-phosphate was isolated in the laboratory from ribonuclease digests Jf ribonucleic acid as described by Markham and Smith (8). Cytidine .V,d'-phosphate and adenosine B',S'-phosphate were synthesized chemically by the method of Brown, Magrath, and Todd (9). Guanosine V,S'-phosphate was isolated from ribonuclease digests of ribonucleic acid as described by Markham and Smith (8) and modified by Heppel, Whitfeld and Markham (2). The dinucleotides were isolated from ribonuclease digests of yeast ribonucleic acid by ion-exchange chromatography (10).
2002
Data are reported for T m , the temperature midpoint of the thermal unfolding curve, of ribonuclease A, versus pH (range 2-9) and salt concentration (range 0-1 M) for two salts, Na 2 SO 4 and NaCl. The results show stabilization by sulfate via anion-specific binding in the concentration range 0-0.1 M and via the Hofmeister effect in the concentration range 0.1-1.0 M. The increase in T m caused by anion binding at 0.1 M sulfate is 20°at pH 2 but only 1°at pH 9, where the net proton charge on the protein is near 0. The 10°increase in T m between 0.1 and 1.0 M Na 2 SO 4 , caused by the Hofmeister effect, is independent of pH. A striking property of the NaCl results is the absence of any significant stabilization by 0.1 M NaCl, which indicates that any Debye screening is small. pH-dependent stabilization is produced by 1 M NaCl: the increase in T m between 0 and 1.0 M is 14°at pH 2 but only 1°at pH 9. The 14°increase at pH 2 may result from anion binding or from both binding and Debye screening. Taken together, the results for Na 2 SO 4 and NaCl show that native ribonuclease A is stabilized at low pH in the same manner as molten globule forms of cytochrome c and apomyoglobin, which are stabilized at low pH by low concentrations of sulfate but only by high concentrations of chloride. .
Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme
Proceedings of the National Academy of Sciences, 2010
Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK a ¼ 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5′ oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolatemodified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2′-O nucleophile by G638 and protonation of the 5′-O leaving group by A756.
Contribution of active site residues to the activity and thermal stability of ribonuclease Sa
Protein Science, 2003
We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k cat is reduced sixfold despite the fact that Glu 74 is over 15 Å from the active site. The pH dependence of k cat /K M is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.