A comparative study on the catalytic properties of guanyl‐specific ribonucleases

Ribonuclease A (RNase A) can make multiple contacts with an RNA substrate. In particular, the enzymatic active site and adjacent subsites bind sequential phosphoryl groups in the RNA backbone through Coulombic interactions. Here, oligomers of vinylsulfonic acid (OVS) are shown to be potent inhibitors of RNase A that exploit these interactions. Inhibition is competitive with substrate and has K i ‫؍‬ 11 pM in assays at low salt concentration. The effect of salt concentration on inhibition indicates that nearly eight favorable Coulombic interactions occur in the RNase A⅐OVS complex. The phosphonic acid and sulfuric acid analogs of OVS are also potent inhibitors although slightly less effective. OVS is also shown to be a contaminant of MES and other buffers that contain sulfonylethyl groups. Oligomers greater than nine units in length can be isolated from commercial MES buffer. Inhibition by contaminating OVS is responsible for the apparent decrease in catalytic activity that has been observed in assays of RNase A at low salt concentration. Thus, OVS is both a useful inhibitor of RNase A and a potential bane to chemists and biochemists who use ethanesulfonic acid buffers.

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“…Ϫ1 at 268 nm (14). The cleavage of poly(C) was monitored by the decrease in ultraviolet hypochromicity.…”

Section: Methodsmentioning

confidence: 99%

Potent Inhibition of Ribonuclease A by Oligo(vinylsulfonic Acid)

Smith

Soellner

Raines

2003

Journal of Biological Chemistry

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“…Ϫ1 per nucleotide at 268 nm (23). Assays were performed at 25°C in 0.10 M MES-NaOH buffer, pH 6.0, containing NaCl (0.10 M), poly(C) (30 M Ϫ1.6 mM), and enzyme (0.75 pM Ϫ3.1 nM).…”

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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“…The cleavage of poly(C) was monitored at 238 nm. The Δε at 250 nm, as calculated from the difference in molar absorptivity of the polymeric substrate and the mononucleotide cyclic phosphate product, has been reported to be 2380 M −1 cm −1 at pH 6.2 (51). The Δε at 238 nm was determined to be 2792 M −1 cm −1 by observing the change in absorption of a partially cleaved substrate at 250 nm and 238 nm.…”

Section: Enzyme Kineticsmentioning

confidence: 99%

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

1998

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The sidechains of histidine and aspartate residues form a hydrogen bond in the active sites of many enzymes. In serine proteases, the His⋯Asp hydrogen bond of the catalytic triad is known to contribute greatly to catalysis, perhaps via the formation of a low-barrier hydrogen bond. In bovine pancreatic ribonuclease A (RNase A), the His⋯Asp dyad is composed of His119 and Asp121. Previously, sitedirected mutagenesis was used to show that His119 has a fundamental role-to act as an acid during catalysis of RNA cleavage , J. Am. Chem. Soc. 116,[5467][5468]. Here, Asp121 was replaced with an asparagine or alanine residue. The crystalline structures of the two variants were determined by X-ray diffraction analysis to a resolution of 1.6 Å with an R-factor of 0.18. Replacing Asp121 with an asparagine or alanine residue does not perturb the overall conformation of the enzyme. In the structure of D121N RNase A, N δ rather than O δ of Asn121 faces His119. This alignment in the crystalline state is unlikely to exist in solution because catalysis by the D121N variant is not compromised severely. The steady-state kinetic parameters for catalysis by the wild-type and variant enzymes were determined for the cleavage of uridylyl(3′→5′)adenosine and poly(cytidylic acid), and for the hydrolysis of uridine 2′,3′-cyclic phosphate. Replacing Asp121 decreases the values of k cat /K m and k cat for cleavage by 101-fold (D121N) and 10 2 -fold (D121A). Replacing Asp121 also decreases the values of k cat /K m and k cat for hydrolysis by 10 0.5 -fold (D121N) and 10-fold (D121A), but has no other effect on the pH-rate profiles for hydrolysis. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. Apparently, the major role of Asp121 is to orient the proper tautomer of His119 for catalysis. Thus, the mere presence of a His⋯Asp dyad in an enzymic active site is not a mandate for its being crucial in effecting catalysis.Keywords catalytic dyad; catalytic triad; low-barrier hydrogen bond; pH-rate profile; ribonuclease A; serine protease; site-directed mutagenesis; X-ray crystallographyThe amino acid motifs available to enzymic catalysts are diverse. Still, many enzymes fall into classes wherein great similarities exist. One well-studied class is that of the serine proteases (1). This class of enzymes has an active-site motif known as the catalytic triad, which is composed of the residues Ser⋯His⋯Asp linked by hydrogen bonds (2,3). † This work was supported by Grant GM44783 (NIH). X-ray data collection and computational facilities were supported by Grant BIR-9317398 (NSF). L.W.S. was supported by postdoctoral fellowship CA69750 (NIH). D.J.Q. was supported by Cellular and Molecular Biology Training Grant GM07215 (NIH).*Author to whom correspondence should be addressed.. ‡ Present address: School of Pharmacy, University of Wisconsin -Madison, Madison, WI 53706. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2...

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A comparative study on the catalytic properties of guanyl‐specific ribonucleases

Cited by 22 publications

References 18 publications

Potent Inhibition of Ribonuclease A by Oligo(vinylsulfonic Acid)

Potent Inhibition of Ribonuclease A by Oligo(vinylsulfonic Acid)

A New Remote Subsite in Ribonuclease A

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

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