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1.
Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog) 总被引:3,自引:0,他引:3
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Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, ~50 and ~25%, respectively. 相似文献
2.
Onconase (ONC), a homologue of ribonuclease A (RNase A), is in clinical trials for the treatment of cancer. ONC possesses a conserved active-site catalytic triad, which is composed of His10, Lys31, and His97. The three-dimensional structure of ONC suggests that two additional residues, Lys9 and an N-terminal lactam formed from a glutamine residue (Pca1), could also contribute to catalysis. To determine the role of Pca1, Lys9, and Lys31 in the function of ONC, site-directed mutagenesis was used to replace each with alanine. Values of k(cat)/K(M) for the variants were determined with a novel fluorogenic substrate, which was designed to match the nucleobase specificity of ONC and gives the highest known k(cat)/K(M) value for the enzyme. The K9A and K31A variants display 10(3)-fold lower k(cat)/K(M) values than the wild-type enzyme, and a K9A/K31A double variant suffers a >10(4)-fold decrease in catalytic activity. In addition, replacing Lys9 or Lys31 eliminates the antitumoral activity of ONC. The side chains of Pca1 and Lys9 form a hydrogen bond in crystalline ONC. Replacing Pca1 with an alanine residue lowers the catalytic activity of ONC by 20-fold. Yet, replacing Pca1 in the K9A variant enzyme does not further reduce catalytic activity, revealing that the function of the N-terminal pyroglutamate residue is to secure Lys9. The thermodynamic cycle derived from k(cat)/K(M) values indicates that the Pca1...Lys9 hydrogen bond contributes 2.0 kcal/mol to the stabilization of the rate-limiting transition state during catalysis. Finally, binding isotherms with a substrate analogue indicate that Lys9 and Lys31 contribute little to substrate binding and that the low intrinsic catalytic activity of ONC originates largely from the low affinity of the enzyme for its substrate. These findings could assist the further development of ONC as a cancer chemotherapeutic. 相似文献
3.
Many proteins and bioactive peptides contain an N-terminal pyroglutamate residue (Pyr1). This residue reduces the susceptibility of the protein to aminopeptidases and often has important functional roles. The antitumor ribonuclease RC-RNase 3 (RNase 3) from oocytes of Rana catesbeiana (bullfrog) is one such protein. We have produced recombinant RNase 3 containing the N-terminal Pyr1 (pRNase 3) and found it to be indistinguishable from the native RNase 3 by mass spectrometry and a variety of other biochemical and immunological criteria. We demonstrated by NMR analysis that the Pyr1 of pRNase 3 forms hydrogen bonds with Lys9 and Ile96 and stabilizes the N-terminal alpha-helix in a rigid conformation. In contrast, the N-terminal alpha-helix becomes flexible and the pKa values of the catalytic residues His10 and His97 altered when Pyr1 formation is blocked by an extra methionine at the N terminus in the recombinant mqRNase 3. Thus, our results provide a mechanistic explanation on the essential role of Pyr1 in maintaining the structural integrity, especially at the N-terminal alpha-helix, and in providing the proper environment for the ionization of His10 and His97 residues for catalysis and cytotoxicity against HeLa cells. 相似文献
4.
Marcia C Haigis Erin L Kurten Richele L Abel Ronald T Raines 《The Journal of biological chemistry》2002,277(13):11576-11581
Onconase(ONC) is an amphibian ribonuclease that is in clinical trials as a cancer chemotherapeutic agent. ONC is a homolog of ribonuclease A (RNase A). RNase A can be made toxic to cancer cells by replacing Gly(88) with an arginine residue, thereby enabling the enzyme to evade the endogenous cytosolic ribonuclease inhibitor protein (RI). Unlike ONC, RNase A contains a KFERQ sequence (residues 7-11), which signals for lysosomal degradation. Here, substitution of Arg(10) of the KFERQ sequence has no effect on either the cytotoxicity of G88R RNase A or its affinity for RI. In contrast, K7A/G88R RNase A is nearly 10-fold more cytotoxic than G88R RNase A and has more than 10-fold less affinity for RI. Up-regulation of the KFERQ-mediated lysosomal degradation pathway has no effect on the cytotoxicity of these ribonucleases. Thus, KFERQ-mediated degradation does not limit the cytotoxicity of RNase A variants. Moreover, only two amino acid substitutions (K7A and G88R) are shown to endow RNase A with cytotoxic activity that is nearly equal to that of ONC. 相似文献
5.
Onconase (ONC), an amphibian member of the bovine pancreatic ribonuclease A (RNase A) superfamily, is in phase III clinical trials as a treatment for malignant mesothelioma. RNase A is a far more efficient catalyst of RNA cleavage than ONC but is not cytotoxic. The innate ability of ONC to evade the cytosolic ribonuclease inhibitor protein (RI) is likely to be a primary reason for its cytotoxicity. In contrast, the non-covalent interaction between RNase A and RI is one of the strongest known, with the RI.RNase A complex having a K(d) value in the femtomolar range. Here, we report on the use of the fast atomic density evaluation (FADE) algorithm to identify regions in the molecular interface of the RI.RNase A complex that exhibit a high degree of geometric complementarity. Guided by these "knobs" and "holes", we designed variants of RNase A that evade RI. The D38R/R39D/N67R/G88R substitution increased the K(d) value of the pRI.RNase A complex by 20 x 10(6)-fold (to 1.4 microM) with little change to catalytic activity or conformational stability. This and two related variants of RNase A were more toxic to human cancer cells than was ONC. Notably, these cytotoxic variants exerted their toxic activity on cancer cells selectively, and more selectively than did ONC. Substitutions that further diminish affinity for RI (which has a cytosolic concentration of 4 microM) are unlikely to produce a substantial increase in cytotoxic activity. These results demonstrate the utility of the FADE algorithm in the examination of protein-protein interfaces and represent a landmark towards the goal of developing chemotherapeutics based on mammalian ribonucleases. 相似文献
6.
Singh UP Ardelt W Saxena SK Holloway DE Vidunas E Lee HS Saxena A Shogen K Acharya KR 《Journal of molecular biology》2007,371(1):93-111
Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1-4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8-99.1% and 38.2-40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of k(cat)/K(M) with hypersensitive fluorogenic substrates are 10(4) and 10(2)-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B(1) or B(2) subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 A resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual alpha2-beta1 loop may do so) and the B(2) subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107. 相似文献
7.
Onconase((R)) (ONC) is a homolog of ribonuclease A (RNase A) that has unusually high conformational stability and is toxic to human cancer cells in vitro and in vivo. ONC and its amphibian homologs have a C-terminal disulfide bond, which is absent in RNase A. Replacing this cystine with a pair of alanine residues greatly decreases the conformational stability of ONC. In addition, the C87A/C104A variant is 10-fold less toxic to human leukemia cells. These data indicate that the synapomorphic disulfide bond of ONC is an important determinant of its cytotoxicity. 相似文献
8.
Onconase (ONC) from Rana pipiens is the smallest member of the ribonuclease A (RNase A) superfamily. Despite a tertiary structure similar to RNase A, ONC is distinguished by an extremely high thermodynamic stability. In the present paper we have probed the significance of three structural regions, which exhibit structural peculiarities in comparison to RNase A, for the stability of ONC to temperature and guanidine hydrochloride induced denaturation: (i) the N-terminal pyroglutamate residue, (ii) the hydrophobic cluster between helix I and the first beta-sheet, and (iii) the C-terminal disulfide bond. For this purpose, the enzyme variants 相似文献
9.
Matousek J Soucek J Slavík T Tománek M Lee JE Raines RT 《Comparative biochemistry and physiology. Toxicology & pharmacology : CBP》2003,136(4):343-356
Onconase (ONC) and bovine seminal ribonuclease (BS-RNase) are homologs of bovine pancreatic ribonuclease (RNase A). Unlike RNase A, ONC and BS-RNase can evade the cytosolic ribonuclease inhibitor protein and are potent cytotoxins. Here, the endogenous cytotoxic activities of ONC and BS-RNase are compared in a wide variety of assays. Injections of ONC into one or both testes of mice and rats evokes a stronger aspermatogenic activity than does the injection of BS-RNase. Epididymides exposed to ONC lose mass and all sperm. Testicular tissue is gradually colonized by immunite and fibrocytic cells. Yet, Leydig cells are always present and functional in the ligamented parts of testicles injected with ONC or BS-RNase. ONC is likewise more toxic to mouse embryos than is BS-RNase, both in vitro and in vivo. The antiproliferative effect of ONC on human tumor cell line ML-2 and lymphocytes in a mixed lymphocyte culture is also more pronounced than is that of BS-RNase. The number of granulocyte-macrophage colony-forming units is repressed almost completely by ONC, whereas a five-fold higher dose of BS-RNase does not cause substantial inhibition. In mice, ONC is less immunogenic than BS-RNase but more immunogenic than RNase A. Together, these data indicate that ONC is a pluripotent cytotoxin, and serves as the benchmark with which to gauge the cytotoxicity of other ribonucleases. 相似文献
10.
Leu YJ Chern SS Wang SC Hsiao YY Amiraslanov I Liaw YC Liao YD 《The Journal of biological chemistry》2003,278(9):7300-7309
The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A superfamily, exert cytotoxicity toward tumor cells. RC-RNase, the most active among frog ribonucleases, has a unique base preference for pyrimidine-guanine rather than pyrimidine-adenine in RNase A. Residues of RC-RNase involved in base specificity and catalytic activity were determined by site-directed mutagenesis, k(cat)/K(m) analysis toward dinucleotides, and cleavage site analysis of RNA substrate. The results show that Pyr-1 (N-terminal pyroglutamate), Lys-9, and Asn-38 along with His-10, Lys-35, and His-103 are involved in catalytic activity, whereas Pyr-1, Thr-39, Thr-70, Lys-95, and Glu-97 are involved in base specificity. The cytotoxicity of RC-RNase is correlated, but not proportional to, its catalytic activity. The crystal structure of the RC-RNase.d(ACGA) complex was determined at 1.80 A resolution. Residues Lys-9, His-10, Lys-35, and His-103 interacted directly with catalytic phosphate at the P(1) site, and Lys-9 was stabilized by hydrogen bonds contributed by Pyr-1, Tyr-28, and Asn-38. Thr-70 acts as a hydrogen bond donor for cytosine through Thr-39 and determines B(1) base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form four hydrogen bonds with guanine at B(2) site and determine B(2) base specificity. 相似文献
11.
Mallorquí-Fernández G Pous J Peracaula R Aymamí J Maeda T Tada H Yamada H Seno M de Llorens R Gomis-Rüth FX Coll M 《Journal of molecular biology》2000,300(5):1297-1307
Eosinophil cationic protein (ECP; RNase 3) is a human ribonuclease found only in eosinophil leukocytes that belongs to the RNase A superfamily. This enzyme is bactericidal, helminthotoxic and cytotoxic to mammalian cells and tissues. The protein has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data up to 1. 75 A resolution. The molecule displays the alpha+beta folding topology typical for members of the ribonuclease A superfamily. The catalytic active site residues are conserved with respect to other ribonucleases of the superfamily but some differences appear at substrate recognition subsites, which may account, in part, for the low catalytic activity. Most strikingly, 19 surface-located arginine residues confer a strong basic character to the protein. The high concentration of positive charges and the particular orientation of the side-chains of these residues may also be related to the low activity of ECP as a ribonuclease and provides an explanation for its unique cytotoxic role through cell membrane disruption. 相似文献
12.
Ian W. Windsor Crystal J. Graff Ronald T. Raines 《Protein science : a publication of the Protein Society》2019,28(9):1713-1719
The endogenous production of enzymes as zymogens provides a means to control catalytic activities. Here, we describe the heterologous production of ribonuclease 1 (RNase 1), which is the most prevalent secretory ribonuclease in humans, as a zymogen. In folded RNase 1, the N and C termini flank the enzymic active site. By using intein‐mediated cis‐splicing, we created circular proteins in which access to the active site of RNase 1 is obstructed by an amino‐acid sequence that is recognized by the HIV‐1 protease. Installing a sequence that does not perturb the RNase 1 fold led to only modest inactivation. In contrast, the ancillary truncation of residues from each terminus led to a substantial decrease in the catalytic activity of the zymogen with the maintenance of thermostability. For optimized zymogens, activation by HIV‐1 protease led to a > 104‐fold increase in ribonucleolytic activity at a rate comparable to that for the cleavage of endogenous viral substrates. Molecular modeling indicated that these zymogens are inactivated by conformational distortion in addition to substrate occlusion. Because protease levels are elevated in many disease states and ribonucleolytic activity can be cytotoxic, RNase 1 zymogens have potential as generalizable prodrugs. 相似文献
13.
A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbeiana (bullfrog) oocytes. 总被引:1,自引:0,他引:1
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Y D Liao 《Nucleic acids research》1992,20(6):1371-1377
A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS. 相似文献
14.
Donald Gagné Laurie-Anne Charest Sébastien Morin Evgenii L. Kovrigin Nicolas Doucet 《The Journal of biological chemistry》2012,287(53):44289-44300
Conformational flexibility between structural ensembles is an essential component of enzyme function. Although the broad dynamical landscape of proteins is known to promote a number of functional events on multiple time scales, it is yet unknown whether structural and functional enzyme homologues rely on the same concerted residue motions to perform their catalytic function. It is hypothesized that networks of contiguous and flexible residue motions occurring on the biologically relevant millisecond time scale evolved to promote and/or preserve optimal enzyme catalysis. In this study, we use a combination of NMR relaxation dispersion, model-free analysis, and ligand titration experiments to successfully capture and compare the role of conformational flexibility between two structural homologues of the pancreatic ribonuclease family: RNase A and eosinophil cationic protein (or RNase 3). In addition to conserving the same catalytic residues and structural fold, both homologues show similar yet functionally distinct clusters of millisecond dynamics, suggesting that conformational flexibility can be conserved among analogous protein folds displaying low sequence identity. Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins. These results support the hypothesis that conformational flexibility is partly required for catalytic function in homologous enzyme folds, further highlighting the importance of dynamic residue sectors in the structural organization of proteins. 相似文献
15.
Synergistic cytotoxicity of Rana catesbeiana ribonuclease and IFN-gamma on hepatoma cells 总被引:3,自引:0,他引:3
Hu CC Lee YH Tang CH Cheng JT Wang JJ 《Biochemical and biophysical research communications》2001,280(5):1229-1236
RC-RNase purified from Rana catesbeiana (bullfrog) oocytes is a pyrimidine-guanine sequence-specific ribonuclease. RC-RNase is derived from the RNase superfamily genes exerting distinct ribonucleolytic activity and possesses cytotoxicity to tumor cells, but rarely to primary cells. In this study, we utilized RC-RNase to function with antiproliferative cytokines. The combination with TNF-alpha or TNF-beta would not aggravate cell death. However, the combination with IFN-gamma could induce synergistic cytotoxicity verified by XTT assays toward three hepatoma cell lines bearing different differentiation stages. The distinct cytotoxicity from RC-RNase or RC-RNase/IFN-gamma on different hepatoma cells was correlated with the differentiation extent but not the proliferation rate of the cells. Despite the synergistic cytotoxicity and severe mitochondrial disruptions in the RC-RNase/IFN-gamma-treated cells, we scarcely detected any significant feature of apoptosis or necrosis by FACS analysis on annexin-V/propidium iodide staining. The mechanisms of cell death triggered by RC-RNase or RC-RNase/IFN-gamma require further investigation. 相似文献
16.
Onconasetrade mark, a homolog of bovine pancreatic ribonuclease A (RNase A) with high conformational stability, is cytotoxic and has efficacy as a cancer chemotherapeutic agent. Unlike wild-type RNase A, the G88R variant is toxic to cancer cells. Here, variants in which disulfide bonds were removed from or added to G88R RNase A were used to probe the relationship between conformational stability and cytotoxicity in a methodical manner. The conformational stability of the C40A/G88R/C95A and C65A/C72A/G88R variants is less than that of G88R RNase A. In contrast, a new disulfide bond that links the N and C termini (residues 4 and 118) increases the conformational stability of G88R RNase A and C65A/C72A/G88R RNase A. These changes have little effect on the ribonucleolytic activity of the enzyme or on its ability to evade the cytosolic ribonuclease inhibitor protein. The changes do, however, have a substantial effect on toxicity toward human erythroleukemia cells. Specifically, conformational stability correlates directly with cytotoxicity as well as with resistance to proteolysis. These data indicate that conformational stability is a key determinant of RNase A cytotoxicity and suggest that cytotoxicity relies on avoiding proteolysis. This finding suggests a means to produce new cancer chemotherapeutic agents based on mammalian ribonucleases. 相似文献
17.
Onconase® (ONC) is a homolog of bovine pancreatic ribonuclease (RNase A) from the frog Rana pipiens. ONC displays antitumoral activity and is in advanced clinical trials for the treatment of cancer. Here, we report the first atomic structures of ONC-nucleic acid complexes: a T89N/E91A ONC-5′-AMP complex at 1.65 Å resolution and a wild-type ONC-d(AUGA) complex at 1.90 Å resolution. The latter structure and site-directed mutagenesis were used to reveal the atomic basis for substrate recognition and turnover by ONC. The residues in ONC that are proximal to the scissile phosphodiester bond (His10, Lys31, and His97) and uracil nucleobase (Thr35, Asp67, and Phe98) are conserved from RNase A and serve to generate a similar bell-shaped pH versus kcat/KM profile for RNA cleavage. Glu91 of ONC forms two hydrogen bonds with the guanine nucleobase in d(AUGA), and Thr89 is in close proximity to that nucleobase. Installing a neutral or cationic residue at position 91 or an asparagine residue at position 89 virtually eliminated the 102-fold guanine:adenine preference of ONC. A variant that combined such substitutions, T89N/E91A ONC, actually preferred adenine over guanine. In contrast, installing an arginine residue at position 91 increased the guanine preference and afforded an ONC variant with the highest known kcat/KM value. These data indicate that ONC discriminates between guanine and adenine by using Coulombic interactions and a network of hydrogen bonds. The structure of the ONC-d(AUGA) complex was also used to probe other aspects of catalysis. For example, the T5R substitution, designed to create a favorable Coulombic interaction between ONC and a phosphoryl group in RNA, increased ribonucleolytic activity by twofold. No variant, however, was more toxic to human cancer cells than wild-type ONC. Together, these findings provide a cynosure for understanding catalysis of RNA cleavage in a system of high medicinal relevance. 相似文献
18.
The crystal structure of ribonuclease HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI) was determined at 1.6 A resolution. Sto-RNase HI exhibits not only RNase H activity but also double-stranded RNA-dependent ribonuclease (dsRNase) activity. The main-chain fold and steric configurations of the four acidic active-site residues of Sto-RNase HI are very similar to those of other type 1 RNases H. However, Arg118 of Sto-RNase HI is located at the position in which His124 of E. coli RNase HI, His539 of HIV-1 RNase H, and Glu188 of Bacillus halodurans RNase H are located. The mutation of this residue to Ala considerably reduced both the RNase H and dsRNase activities without seriously affecting substrate binding, suggesting that Arg118 is involved in catalytic function. This residue may promote product release by perturbing the coordination of the metal ion A as proposed for Glu188 of B. halodurans RNase H. In addition, the extreme C-terminal region of Sto-RNase HI is anchored to its core region by one disulfide bond and several hydrogen bonds. Differential scanning calorimetry measurements indicated that Sto-RNase HI is a hyperstable protein with a melting temperature of 102 degrees C. The mutations of the cysteine residues forming disulfide bond or elimination of the extreme C-terminal region greatly destabilized the protein, indicating that anchoring of the C-terminal tail is responsible for hyperstabilization of Sto-RNase HI. 相似文献
19.
Onconase(?) (ONC) is an amphibian member of the pancreatic ribonuclease superfamily that is selectively toxic to tumor cells. It is a much less efficient enzyme than the archetypal ribonuclease A and, in an attempt to gain further insight, we report the first atomic resolution crystal structure of ONC, determined in complex with sulfate ions at 100 K. The electron density map is of a quality sufficient to reveal significant nonplanarity in several peptide bonds. The majority of active site residues are very well defined, with the exceptions being Lys31 from the catalytic triad and Lys33 from the B(1) subsite, which are relatively mobile but rigidify upon nucleotide binding. Cryocooling causes a compaction of the unit cell and the protein contained within. This is principally the result of an inward movement of one of the lobes of the enzyme (lobe 2), which also narrows the active site cleft. Binding a nucleotide in place of sulfate is associated with an approximately perpendicular movement of lobe 2 and has little further effect on the cleft width. Aspects of this deformation are present in the principal axes of anisotropy extracted from C(α) atomic displacement parameters, indicating its intrinsic nature. The three lowest-frequency modes of ONC motion predicted by an anisotropic network model are compaction/expansion variations in which lobe 2 is the prime mover. Two of these have high similarity to the cryocooling response and imply that the essential 'breathing' motion of ribonuclease A is conserved in ONC. Instead, shifts in conformational equilibria may contribute to the reduced ribonucleolytic activity of ONC. 相似文献
20.
The motion of amino acid residues on the millisecond (ms) time scale is involved in the tight regulation of catalytic function in numerous enzyme systems. Using a combination of mutational, enzymological, and relaxation-compensated (15)N Carr-Purcell-Meiboom-Gill (CPMG) methods, we have previously established the conformational significance of the distant His48 residue and the neighboring loop 1 in RNase A function. These studies suggested that RNase A relies on an intricate network of hydrogen bonding interactions involved in propagating functionally relevant, long-range ms motions to the catalytic site of the enzyme. To further investigate the dynamic importance of this H-bonding network, this study focuses on the individual replacement of Thr17 and Thr82 with alanine, effectively altering the key H-bonding interactions that connect loop 1 and His48 to the rest of the protein. (15)N CPMG dispersion studies, nuclear magnetic resonance (NMR) chemical shift analysis, and NMR line shape analysis of point mutants T17A and T82A demonstrate that the evolutionarily conserved single H-bond linking His48 to Thr82 is essential for propagating ms motions from His48 to the active site of RNase A on the time scale of catalytic turnover, whereas the T17A mutation increases the off rate and conformational exchange motions in loop 1. Accumulating evidence from our mutational studies indicates that residues experiencing conformational exchange in RNase A can be grouped into two separate clusters displaying distinct dynamical features, which appear to be independently affected by mutation. Overall, this study illuminates how tightly controlled and finely tuned ms motions are in RNase A, suggesting that designed modulation of protein motions may be possible. 相似文献