Contribution of a tyrosine side chain to ribonuclease A catalysis and stability–Contribution of Tyr 97 to RNase A catalysis and stability

Eberhardt, Eric S.; Wittmayer, Paula K.; Templer, Barbra M.; Raines, Ronald T.

doi:10.1002/pro.5560050823

Cited by 39 publications

(40 citation statements)

References 46 publications

(32 reference statements)

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“…Interestingly, this hydrogen bond is also observed in ribonuclease A (corresponding residues Lys41 and Tyr97) where the hydroxylic proton is slowly exchanging with solvent as well . This interaction has been shown to be strictly conserved in all known structures of the pancreatic ribonuclease superfamily and seems to be important for proper orientation of the Lys within the active site (Eberhardt et al, 1996). The side-chain indeed adopts an extended conformation and is oriented towards the active-site His13.…”

Section: ~M K K K G I T S P C K D I N T F I H G N K R S ~K A~c E N K mentioning

confidence: 87%

Three‐Dimensional Solution Structure of Human Angiogenin Determined by ¹H,¹⁵N‐NMR Spectroscopy — Characterisation of Histidine Protonation States and Pk_a Values

Lequin

Thüring

Robin³

et al. 1997

European Journal of Biochemistry

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Human angiogenin is a member of the pancreatic ribonuclease superfamily that induces blood vessel foimation. Its three-dimensional solution structure has been determined to high resolution by heteronuclear NMR spectroscopy. 30 structures were calculated, based on a total of 1441 assigned NOE correlations, 64 coupling constants and 50 hydrogen bonds. The backbone atomic rms difference from the mean coordinates is 0.067 ? 0.012 nm and 0.13 nm from the previously determined crystal structure. The sidechain of Gln117 was found to obstruct the active site as observed in the crystal state. There was no evidence of an alternative open form of angiogenin, although two sets of chemical shifts were observed for some residues, mainly around the active site and in the C-terminal segment. The topology of the ribonucleolytic active site is described with a particular emphasis on the conformation and protonation of active-site His residues. The side-chain of His114 adopts two main conformations in solution. In contrast to pancreatic ribonuclease A, His13 was shown to be more basic than Hisl14, with pK, values of 6.65 and 6.05 respectively. The His47 residue is located in an environment very resistant to protonation with a pK, lower than 4.

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Section: ~M K K K G I T S P C K D I N T F I H G N K R S ~K A~c E N K mentioning

confidence: 87%

Three‐Dimensional Solution Structure of Human Angiogenin Determined by ¹H,¹⁵N‐NMR Spectroscopy — Characterisation of Histidine Protonation States and Pk_a Values

Lequin

Thüring

Robin³

et al. 1997

European Journal of Biochemistry

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“…Because cytotoxicity is assessed by prolonged incubation of ribonucleases at physiological temperature, it is important to establish that the RNase A variants retain adequate thermal stability. Thermal stabilities of the variants were measured by monitoring the change in absorbance at 287 nm (A 287 ) with increasing temperature (42). The temperature of the ribonuclease solutions (0.1-0.2 mg͞ml in PBS) was increased from 25°C to 80°C in 1°C increments.…”

Section: Methodsmentioning

confidence: 99%

Ribonuclease A variants with potent cytotoxic activity

Leland

Schultz

Kim

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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206

304

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Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI-RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.

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“…Thermal Denaturation-Stabilities of the wild-type and variant proteins were determined by thermal denaturation studies in 0.10 M MESNaOH buffer, pH 6.0, as described elsewhere (22).…”

Section: Methodsmentioning

confidence: 99%

A New Remote Subsite in Ribonuclease A

Fisher

Grilley²,

Raines

1998

Journal of Biological Chemistry

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The interaction between bovine pancreatic ribonuclease A (RNase A) and its RNA substrate extends beyond the scissile bond. Enzymic subsites interact with the bases and the phosphoryl groups of a bound substrate. We evaluated the four cationic residues closest to known subsites for their abilities to interact with a bound nucleic acid. Lys-37, Arg-39, Arg-85, and Lys-104 were replaced individually by an alanine residue, and the resulting enzymes were assayed as catalysts of poly-(cytidylic acid) (poly(C)) cleavage. The values of K m and k cat /K m for poly(C) cleavage were affected only by replacing Arg-85. Moreover, the contribution of Arg-85 to the binding of the ground state and the transition state was uniformOK m increased by 15-fold and k cat /K m decreased by 10-fold. The contribution of Arg-85 to binding was also apparent in the values of K d for complexes with oligonucleotides of different length. This contribution was dependent on salt concentration, as expected from a coulombic interaction between a cationic side chain and an anionic phosphoryl group. Together, these data indicate that Arg-85 interacts with a particular phosphoryl group of a bound nucleic acid. We propose that Arg-85 comprises a new distal subsite in RNase AOthe P(؊1) subsite.The efficiency of enzymatic catalysts is a source of continued interest and inspiration as molecular scientists strive to design new catalysts. A distinguishing characteristic of enzymic catalysts is that they bind to their substrates (1, 2). Binding energy is necessary to compensate for the loss of translational and rotational entropy and for any destabilization of the substrate required to reach the transition state (3, 4). Multivalent contacts between an enzyme and substrate provide much of this required binding energy. Indeed, many enzymes that cleave polymeric substrates have subsites that interact with monomeric units of the substrate.Bovine pancreatic ribonuclease A (RNase A; 1 EC 3.1.27.5) is a classic model for revealing the physical, chemical, and biological properties of enzymes (5, 6). RNase A is a 13.7-kDa endoribonuclease that binds RNA in a cationic cleft and cleaves on the 3Ј-side of pyrimidine residues. The cleft contains subsites (B1, B2, and B3) that interact specifically with bases and subsites (P0, P1, and P2) that interact with phosphoryl groups (7,8). The specificity of RNase A for pyrimidine bases is because of exclusion of the larger purine bases from the B1 subsite (9). The B2 and B3 subsites prefer to bind purine bases. His-12, His-119, and Lys-41 of the P1 subsite are the residues most central to the catalytic function of the enzyme. The amino acid residues that comprise the P0 (Lys-66) and P2 (Lys-7 and Arg-10) subsites increase the affinity with which the substrate binds to the enzyme and participate indirectly in catalysis (10 -12).Some data portend the existence of additional RNase A binding sites beyond those characterized previously. Three-dimensional structures derived from x-ray diffraction analyses reveal a line of cationic res...

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Contribution of a tyrosine side chain to ribonuclease A catalysis and stability–Contribution of Tyr 97 to RNase A catalysis and stability

Cited by 39 publications

References 46 publications

Three‐Dimensional Solution Structure of Human Angiogenin Determined by ¹H,¹⁵N‐NMR Spectroscopy — Characterisation of Histidine Protonation States and Pk_a Values

Three‐Dimensional Solution Structure of Human Angiogenin Determined by ¹H,¹⁵N‐NMR Spectroscopy — Characterisation of Histidine Protonation States and Pk_a Values

Ribonuclease A variants with potent cytotoxic activity

A New Remote Subsite in Ribonuclease A

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Contribution of a tyrosine side chain to ribonuclease A catalysis and stability–Contribution of Tyr 97 to RNase A catalysis and stability

Cited by 39 publications

References 46 publications

Three‐Dimensional Solution Structure of Human Angiogenin Determined by 1H,15N‐NMR Spectroscopy — Characterisation of Histidine Protonation States and Pka Values

Three‐Dimensional Solution Structure of Human Angiogenin Determined by 1H,15N‐NMR Spectroscopy — Characterisation of Histidine Protonation States and Pka Values

Ribonuclease A variants with potent cytotoxic activity

A New Remote Subsite in Ribonuclease A

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Product

Resources

About

Three‐Dimensional Solution Structure of Human Angiogenin Determined by ¹H,¹⁵N‐NMR Spectroscopy — Characterisation of Histidine Protonation States and Pk_a Values

Three‐Dimensional Solution Structure of Human Angiogenin Determined by ¹H,¹⁵N‐NMR Spectroscopy — Characterisation of Histidine Protonation States and Pk_a Values