A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

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 3'-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.     

Chemical modification by pyridoxal 5''-phosphate and cyclohexane-1,2-dione indicates that Lys-7 and Arg-10 are involved in the p2 phosphate-binding subsite of bovine pancreatic ribonuclease A.

Steric and chemical evidence had previously shown that residues Lys-7 and/or Arg-10 of bovine pancreatic RNAase A could belong to the p2 phosphate-binding subsite, adjacent to the 3' side of the main site p1. In the present work chemical modification of the enzyme with pyridoxal 5'-phosphate and cyclohexane-1,2-dione was carried out in order to identify these residues positively as part of the p2 site. The reaction with pyridoxal 5'-phosphate yields three monosubstituted derivatives, at Lys-1, Lys-7 and Lys-41. A strong decrease in the yield of derivatives at Lys-7 and Lys-41 was observed when either p1 or p2 was specifically blocked by 5'-AMP or 3'-AMP respectively. These experiments indicate that both sites are needed for the reaction of pyridoxal 5'-phosphate with RNAase A to take place. The positive charge in one of the sites interacts with the phosphate group of pyridoxal 5'-phosphate, giving the proper orientation to the carbonyl group, which then reacts with the lysine residue present in the other site. The absence of reaction between pyridoxal 5'-phosphate and an RNAase derivative that has the p2 site blocked supports this hypothesis. Labelling of Lys-7 with pyridoxal 5'-phosphate has a more pronounced effect on the kinetics with RNA than with the smaller substrate 2',3'-cyclic CMP. In addition, when the phosphate moiety of the 5'-phosphopyridoxyl group was removed with alkaline phosphatase the kinetic constants with 2',3'-cyclic CMP returned to values very similar to those of the native enzyme, whereas a higher Km and lower Vmax. were still observed for RNA. This indicates that this new derivative has recovered a free p1 site and, hence, the capability to act on 2',3'-cyclic CMP, but the presence of the pyridoxyl group bound to Lys-7 is still blocking a secondary phosphate-binding site, namely p2. Finally, reaction of cyclohexane-1,2-dione at Arg-10 is suppressed in the presence of 3'-AMP but only a 19% decrease is observed with 5'-AMP, suggesting that Arg-10 is also close to the p2 phosphate-binding subsite.     

The reaction of bovine pancreatic ribonuclease A with 6-chloropurineriboside 5'-monophosphate. Evidence on the existence of a phosphate-binding sub-site

The chemical modification of bovine pancreatic ribonuclease A by 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate was studied under several reaction conditions. The reaction, at pH 7.3, 40 degrees C and a nucleotide: enzyme molar ratio of 60, showed a high degree of specificity in comparison to those corresponding to the base or the nucleoside. The main derivative was isolated by means of CM-cellulose chromatography. Subtilisin cleavage of this derivative showed that the substitution had taken place in the S-peptide moiety. Tryptic digestion of the S-peptide indicated that a lysine residue had been modified. Enzymatic and physico-chemical considerations showed that the actual site of reaction was the alpha-amino group of Lys-1. The structural and kinetic properties of the derivative are consistent with the existence of a phosphate-binding sub-site near the N-terminal region of the enzyme.     

1H-n.m.r. studies on the existence of substrate binding sites in bovine pancreatic ribonuclease A

The reaction of ribonuclease A with either 6-chloropurine riboside 5'-monophosphate or the corresponding nucleoside yields one derivative, with the reagent covalently bound to the alpha-amino group of Lys-1, called derivative II and derivative E, respectively. We studied by means of 1H-n.m.r. at 270 MHz the interaction of these derivatives with different purine ligands. The pK values of His-12- and -119 were obtained and compared with those resulting from the interaction with ribonuclease A. The results showed that the interaction of derivative E with 3'AMP is similar to that described for RNase A as the pK2 of His-12 is increased while that of His-119 remains unaltered. However, derivative II presents some differences as it was found an enhancement of the pK2 values of both His-12 and His-119. Interaction of derivative II and derivative E with dApdA increases the pK2 of His-119, whereas a decrease is found when it interacts with ribonuclease A. These results suggest that the phosphate group and the nucleoside of both derivatives are located in regions of the enzyme where natural substrate analogues have secondary interactions and they can be interpreted as additional binding sites.     

1H-NMR studies on the binding subsites of bovine pancreatic ribonuclease A

The titration curves of the C-2 histidine protons of an RNAase derivative (a covalent derivative obtained by reaction of bovine pancreatic RNAase A (EC 3.1.27.5) with 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate) were studied by means of 1H-NMR spectroscopy at 270 MHz. The interaction of natural (5'AMP, 5'GMP, 5'IMP) and halogenated purine mononucleotides (cl6RMP, br8AMP) with RNAase A was also monitored by using the same technique. The slight change observed in the pK values of the active centre histidine residues of the RNAase derivative, with respect to those in the native enzyme, can be considered as evidence that the phosphate of the label does not interact directly either with His-12 or 119 in the p1 site, but the p2 site as proposed previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571--579). Lys-7 and/or Arg-10 are proposed as part of the p2 phosphate-binding subsite. The pK values of His-12 and 119 and the shift of an aromatic resonance of the native enzyme found on interaction with some purine nucleotides, can be interpreted by postulating that the interaction of 5'AMP, 5'GMP and 5'IMP takes place not only in the so-called purine-binding site B2R2p1 but also in the primary pyrimidine-binding site B1R1 and p0 of RNAase A.     

Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation

Bovine seminal ribonuclease (BS-RNase) contains the MxM (noncovalent dimer) and M=M (free monomer) in constant ratio. The aim of this work was to evaluate the effect of BS-RNase, its monomer and dimer forms, and also various mutants of this enzyme on meiotic completion in cattle oocytes. It was found that BS-RNase has irreversible effects on the meiotic maturation of bovine oocytes in vitro, particularly on the completion of meiosis. The effect of BS-RNase is dose-dependent. In medium supplemented with 1 microg/ml, the results were comparable with those of the control (70% MII oocytes after 24 hr of culture). Whereas 5 microg/ml reduced the number of MII oocytes to 50%, 10 and 25 microg/ml arrested this process completely. The MxM form and RNase A at 5 microg/ml inhibited the maturation rate by 71 and 48%, respectively, but a less significant effect was observed for the M=M form, or the carboxymethylated monomers MCM31 and MCM32 (21%, 16%, and 42% MII oocytes, respectively, in comparison with control). These data demonstrate that bovine ribonucleases can have variable detrimental effects on the maturation of bovine oocyte. J. Exp. Zool. 287:394-399, 2000.     

The properties of the interaction between bovine pancreatic ribonuclease a and mononucleotides supports the existence of several binding sub-sites in the enzyme

The binding of 5'AMP, 5'GMP, 5'CMP, 3'CMP and Cl6RMP to RNAase A was studied by means of the gel filtration technique. It was found that only one molecule of 3'CMP binds strongly to the enzyme although a very unspecific binding is also present. The interaction of 5'AMP and 5'GMP with the enzyme shows one strong binding site and several weak binding sites, whereas two molecules of 5'CMP bind to RNAase A with equal strength. Cl6RMP shows an anomalous behaviour as both split peaks and troughs are found in the chromatogram. The Ka values for 3'CMP and the strong binding site of 5'AMP and 5'GMP are very similar whereas that for the two binding sites of 5'CMP is smaller (about 2.2 X 10(-4)M-1 and 0.5 X 10(-4)M-1, respectively at pH 5.5, I = 0.01 and 25 degrees C). The results are in general agreement with the known multiplicity of ligand-binding subsites in RNAase A.     

Proline isomerization in bovine pancreatic ribonuclease A. 2. Folding conditions

The kinetics of cis-trans isomerization of individual X-Pro peptide groups is used to study the backbone dynamics of bovine pancreatic ribonuclease A (RNase A). We previously developed and validated a fluorescence method for monitoring the cis-trans isomerization of the Tyr92-Pro93 and Asn113-Pro114 peptide groups of RNase A under unfolding conditions [Juminaga, D., Wedemeyer, W. J., and Scheraga, H. A. (1998) Biochemistry 37, 11614-11620]. The essence of this method is to introduce a fluorescent residue (Tyr or Trp) in a position adjacent to the isomerizing proline (if one is not already present) and to eliminate the fluorescence of other such residues adjacent to prolines by mutating them to phenylalanine. Here, we extend this method to observe the cis-trans isomerization of these peptide groups under folding conditions using two site-directed mutants (Y92F and Y115F) of RNase A. Both isomerizations decelerate with increasing concentrations of GdnHCl, with nearly identical m values (1.11 and 1.19 M(-1), respectively) and extrapolated zero-GdnHCl time constants (42 and 32 s, respectively); by contrast, under unfolding conditions, the cis-trans isomerizations of both Pro93 and Pro114 are independent of GdnHCl concentration. Remarkably, the isomerization rates under folding conditions at GdnHCl concentrations above 1 M are significantly slower than those measured under unfolding conditions. The temperature dependence of the Pro114 isomerization under folding conditions is also unusual; whereas Pro93 exhibits an activation energy typical of proline isomerization (19.4 kcal/mol), Pro114 exhibits a sharply reduced activation energy of 5.7 kcal/mol. A structurally plausible model accounts for these results and, in particular, shows that folding conditions strongly accelerate the cis-trans isomerization of both peptide groups to their native cis conformation, suggesting the presence of flickering local structure in their beta-hairpins.     

Expression of bovine pancreatic ribonuclease A in Escherichia coli

A synthetic gene for bovine pancreatic ribonuclease A (RNase A) has been expressed in Escherichia coli as a fusion protein with beta-galactosidase linked by the tetrapeptide Ile-Glu-Gly-Arg. RNase A was cleaved from the fusion using factor Xa, and the resulting product purified and reconstituted. The isolated RNase A was chromatographically, catalytically, and immunologically identical with authentic RNase A. This work argues that the method suggested by Nagai and Thogersen [Nagai, K. & Thogersen, H. C. (1984) Nature (Lond.) 309, 810-812] for releasing fusion proteins is quite general, even when applied to particularly complicated expression problem. The procedure here makes RNase A available for the first time as a model for studying structure-function relationships in proteins using site-directed mutagenesis.     

Kinetic studies on turtle pancreatic ribonuclease: a comparative study of the base specificities of the B2 and P0 sites of bovine pancreatic ribonuclease A and turtle pancreatic ribonuclease

Kinetic constants for the transesterification of eight dinucleoside phosphates CpX and UpX by bovine and turtle pancreatic ribonuclease were determined. Both ribonucleases have a preference for purine nucleotides at the position X. However, bovine ribonuclease, like other mammalian ribonucleases, prefers 6-amino bases at this site, while turtle ribonuclease prefers 6-keto bases. This difference in specificity at the B2 site may be explained by the substitution of glutamic acid at position 111 by valine in turtle ribonuclease. These results have been confirmed by inhibition studies with the four nucleoside triphosphates. Inhibition studies with pT and pTp showed that a cationic binding group (P0) for the 5'-phosphate of the pyrimidine nucleotides bound at the primary B1 site is present in turtle ribonuclease, although lysine at position 66 in bovine ribonuclease is absent in turtle ribonuclease. However, the side chain of lysine 122 in turtle ribonuclease is probably located in the correct position to take over the role as cationic P0 site.     

Isolation and characterization of monodeamidated derivatives of bovine pancreatic ribonuclease A

The isolation and characterization of the initial intermediates formed during the irreversible acid denaturation of enzyme Ribonuclease A are described. The products obtained when RNase A is maintained in 0.5 M HCl at 30 degrees for periods up to 20 h have been analyzed by ion-exchange chromatography on Amberlite XE-64. Four distinct components were found to elute earlier to RNase A; these have been designated RNase Aa2, Aa1c, Aa1b, and Aa1a in order of their elution. With the exception of RNase Aa2, the other components are nearly as active as RNase A. Polyacrylamide gel electrophoresis at near-neutral pH indicated that RNase Aa1a, Aa1b, and Aa1c are monodeamidated derivatives of RNase A; RNase Aa1c contains, in addition, a small amount of a dideamidated component. RNase Aa2, which has 75% enzymic activity as compared to RNase A, consists of dideamidated and higher deamidated derivatives of RNase A. Except for differences in the proteolytic susceptibilities at an elevated temperature or acidic pH, the monodeamidated derivatives were found to have very nearly the same enzymic activity and the compact folded structure as the native enzyme. Fingerprint analyses of the tryptic peptides of monodeamidated derivatives have shown that the deamidations are restricted to an amide cluster in the region 67-74 of the polypeptide chain. The initial acid-catalyzed deamidation occurs in and around the 65-72 disulfide loop giving rise to at least three distinct monodeamidated derivatives of RNase A without an appreciable change in the catalytic activity and conformation of the ribonuclease molecule. Significance of this specific deamidation occurring in highly acidic conditions, and the biological implications of the physiological deamidation reactions of proteins are discussed.     

Activity change during unfolding of bovine pancreatic ribonuclease A in guanidine

Bovine pancreatic ribonuclease A loses almost completely its activity in 2-3 M guanidine, whereas only very slight conformational changes can be detected when following its unfolding by changes in its intrinsic fluorescence at 305 nm and ultraviolet absorbance at 287 nm. Reactivation on diluting out the denaturant is a time-dependent process, indicating that the inactivation is not due to inhibition by a reversible association of the enzyme with guanidine. The kinetic method of following the substrate reaction, in the presence of the denaturant previously proposed for use in the study of rapid inactivation reactions (Tian, W.X. and Tsou, C.-L. (1982) Biochemistry 21, 1028-1032), is applied to examine the inactivation rates of this enzyme during guanidine denaturation, and these have been compared with the unfolding rates as followed by fluorescence and absorbance changes. It is shown that during the unfolding of this enzyme in guanidine, the inactivation of the enzyme occurs within the dead time of mixing in a stopped-flow apparatus and is at least several orders of magnitude faster than the unfolding reaction as detected by the optical parameters. It appears that, as in the case of creatine kinase reported previously, the active site of a small enzyme stabilized by multiple disulfide linkages, such as ribonuclease A, is also situated in a region which is much more liable to being perturbed by denaturants than is the molecule as a whole.     

Modification of bovine pancreatic ribonuclease A with 6-chloropurine riboside

The chemical modification of bovine pancreatic ribonuclease A by 6-chloropurine riboside was studied to obtain information about the role of the purine nucleoside moiety of the ribonucleic acid in the enzyme-substrate interaction. The residues involved in the reaction were identified, after performic acid oxidation and trypsin digestion, by reverse-phase HPLC peptide mapping. The labeled peptides were detected by following the absorbance at 254 nm, and amino acid analyses of these peptides showed that the reaction had taken place with the amino groups of Lys-1, -37, -41, and -91. The specificity of the reaction was unaffected by changing the ligand:protein molar ratio. Partial separation of the reaction products was accomplished by means of chromatography on CM-Sepharose: four labeled fractions corresponding to mono- and bisubstituted derivatives were found. One of the monosubstituted fractions (fraction E) contained a homogeneous protein with the nucleoside bound to the alpha-amino group of Lys-1 whereas the other (fraction D) was a mixture of derivatives labeled in the epsilon-amino group of Lys-1, -37, -41, and -91. Kinetic studies of these two monosubstituted fractions were performed with cytidine 2',3'-phosphate and ribonucleic acid as substrates. These derivatives showed a noncompetitive inhibition-like behavior with respect to RNase A. Results support the existence of several RNase A regions with affinity for purine nucleosides.     

Modification of bovine pancreatic ribonuclease A with the site-specific reagent 4-arsono-2-nitrofluorobenzene. Spectrophotometric titration of arsononitrophenyl ribonuclease A derivatives

The 4-arsono-2-nitrophenyl chromophore can serve as a versatile spectrophotometric probe of the surface structure of proteins. Values of pK1' and pK2' for the arsonic acid ionizations are near 3 and 8, respectively, and the presence of nearby positive and negative charges produces substantial alterations in the spectral response of the probe. Changes in the extinction at the wavelength of maximum difference are 30-50% of the extinction coefficients, epsilonmax, for each ionization of the arsonic acid moiety. The titration of 41-(4-arsono-2-nitrophenyl)ribonuclease A indicates that the arsonate dianion binds near the active-site histidine residues. With protonation of a carboxylate side chain in the acidic region, presumably aspartic acid-121, the active site is disrupted. The 41-(4-arsono-2-nitrophenyl) group interacts to a greater degree with the histidine-119 side chain than it does with the histidine-12 residue. Interactions of uridine or 3'-cytidylic acid with the ligand-binding region of 41-(4-arsono-2-nitrophenyl) ribonuclease A modify the spectrophotometric response extensively. 3'-Cytidylic acid binds 41-(4-arsono-2-nitrophenyl) ribonuclease A with an affinity 300 times less than that for native ribonuclease A and 17 times lower than that for 41-(2,4-dinitrophenyl) ribonuclease A. The arsononitrophenyl chromophore is responsive to changes in the active site of ribonuclease A induced by such perturbants as ligand binding, chemical modification, and both acid and thermal denaturation.