Geometry of the First Step in the Action of Ribonuclease-A

Usher, D. A.; Erenrich, Evelyn S.; Eckstein, Fritz

doi:10.1073/pnas.69.1.115

Cited by 146 publications

(55 citation statements)

References 17 publications

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“…For example, ribonuclease A (30,31) and MuA transposase (4,8) catalyze both transesterification and hydrolysis with inversion of configuration, implying the same mechanism for both reactions. BfiI is the only REase known to date that seems to employ a covalent intermediate.…”

Section: Dna Alcoholysis By Bfiimentioning

confidence: 99%

Site-specific DNA transesterification catalyzed by a restriction enzyme

Sasnauskas¹,

Connolly

Halford

et al. 2007

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

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Most restriction endonucleases use Mg 2؉ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that BfiI, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5 terminus of the cleaved DNA. Under certain conditions, the terminal 3 -OH of one DNA strand can attack the target phosphodiester bond in the other strand to create a DNA hairpin. Transesterification reactions on DNA with phosphorothioate linkages at the target bond proceed with retention of stereoconfiguration at the phosphorus, indicating, uniquely for a restriction enzyme, a twostep mechanism. We propose that BfiI first makes a covalent enzyme-DNA intermediate, and then it resolves it by a nucleophilic attack of water or an alcohol, to yield hydrolysis or transesterification products, respectively.I n the presence of Mg 2ϩ , type II restriction endonucleases (REases) hydrolyze specific phosphodiester bonds in DNA to yield 5Ј-phosphate and 3Ј-hydroxyl termini (1). The stereochemical pathway for phosphodiester hydrolysis by four such enzymes, EcoRI, EcoRV, SfiI, and HpaII (2-4), have been determined. All proceed with inversion of configuration at the scissile phosphate, which implies an odd number of chemical steps in the hydrolytic reaction, and it is most simply accounted for by a direct in-line displacement of the 3Ј-leaving group by water (5-7). High-resolution structures are available for many more REases than have been analyzed stereochemically (1). Their active sites are generally similar, with several carboxylates to act as ligands for Mg 2ϩ in spatially equivalent positions (1). All such enzymes probably act in the same way, by using water to attack the scissile phosphate, to invert its configuration.Endonucleases from the transposase-retroviral integrase family, like MuA or HIV-integrase, catalyze at a single active site two sets of reactions on DNA (4, 8, 9). The first, the endonucleolytic cleavage of the 3Ј end of the transposon or viral DNA, is mechanistically similar to the hydrolysis reactions of REases. The second, termed DNA strand transfer, is a single-step transesterification in which the newly liberated 3Ј-OH group attacks and becomes linked to a phosphate group in the target DNA (9, 10).We ask here whether the BfiI REase can catalyze transesterification reactions in addition to DNA hydrolysis. BfiI cleaves DNA at specified positions downstream from an asymmetric recognition sequence (11). Unlike other REases, it functions without metal ions (12). The N-terminal half of BfiI (13, 14) is similar to Nuc, an EDTA-resistant nuclease from Salmonella typhimurium (15, 16) that belongs to the phospholipase D (PLD) superfamily. The PLD superfamily is a diverse group of proteins that includes phospholipases, phospholipid synthases, bacterial toxins, phosphodiesterases (PDEs), and nucleases (17, 18). The PLD enzymes catal...

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Section: Dna Alcoholysis By Bfiimentioning

confidence: 99%

Site-specific DNA transesterification catalyzed by a restriction enzyme

Sasnauskas¹,

Connolly

Halford

et al. 2007

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

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“…McPherson et al 1980), covalently linked to asparagine 34 (Plummer et al, 1968). The enzyme has been shown to cleave RNA following uridine or cytidine by a 2'-3' cyclic monophosphate intermediate to yield fragments having terminal pyrimidines with 3' phosphate groups (Usher et al, 1972). A number of amino acid residues have been identified at the active site that appear essential for enzymic activity, and these include histidines 12 and 119 and lysine 41.…”

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confidence: 99%

Crystal structure of RNase A complexed with d(pA)4

McPherson

Brayer

Morrison

1986

Journal of Molecular Biology

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Co-crystals of pancreatic RNase A complexed with oligomers of d(pA), were grown from polyethylene glycol4000 at low ionic strength and the X-ray diffraction data were collected to 2.5 A resolution.From a series of heavy-atom derivatives a multiple isomorphous replacement-phased electron density map of the RNase-d(pA), complex was calculated to 3.5 A. By inspection, the disposition of the known structure of RNase in the unit cell was determined and this was confirmed by calculation of a standard crystallographic residual, R. Refinement of the protein alone in the unit cell as a strictly rigid body yielded an R factor of 0.32 at 2.8 A resolution. From difference Fourier syntheses DNA fragments were elucidated and incorporated into a model of the complex. The entire asymmetric unit was refined using a restrained-constrained least-squares procedure (CORELS) interspersed with difference Fourier syntheses. At the present time the crystal structure has been refined to an overall R value of O-215 at 2.5 A resolution.The asymmetric unit of the complex crystals contains four oligomers of d(pA), associated with each molecule of RNase. In addition, there may also be partially ordered fragments of DNA at low occupancy present in the unit cell, but these have not, at this time, been incorporated into the model: One tetramer of d(pA), is entirely bound by a single protein molecule and occupies a portion of the active site cleft, filling the purine binding site and the phosphate site at the catalytic center with its 5' nucleotide. Two other tetramers are partly intermolecular. One passes from near the pyrimidine binding site over the surface of the protein toward arginine 39 and into a solvent region. A third tetramer is anchored at its 5' terminus by a salt link to lysine 98, passes near arginine 39 and then through a solvent region to terminate with its 3' end near the surface of another protein molecule in the lattice. The fourth tetramer of d(pA), is bound at its 5' end on the opposite side of the protein from the active site in an electropositive anion trap that includes lysines 31 and 91 as well as arginine 33. There may be a DNA-DNA interaction involving the 5' phosphate of one tetramer and the 3' bases of two other tetramers and this may help to stabilize the crystalline complex.If t'he sites of interaction between the protein and the d(pA), fragments are mapped on the surface of the protein, they describe a nearly continuous path into and through the active site, across the surface of the enzyme and finally into the basic amino acid cluster on the opposite side of the protein. Such a virtual DNA strand could explain the observation that, when RNase binds to a long single strand of nucleic acid, it can cover or protect 11 to 12 bases along the polynycleotide chain. The path would also account for the observation that at least seven individual electrostatic interactions are involved in the binding of RNase to single-stranded nucleic acids.

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“…1). The nucleophilic attack occurs inline, in a postulated trigonal bipyramidal transition state, implying a catalytic base and acid on either side of the scissile bond (1,2). X-ray crystallographical and site-directed mutagenesis experiments have provided substantial insight in the structure-function relationship of two unrelated families of ribonucleases.…”

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confidence: 99%

A Nucleophile Activation Dyad in Ribonucleases

Mignon¹,

Steyaert²,

Loris³

et al. 2002

Journal of Biological Chemistry

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Ribonucleases (RNases) catalyze the cleavage of the phosphodiester bond in RNA up to 10 15 -fold, as compared with the uncatalyzed reaction. High resolution crystal structures of these enzymes in complex with 3-mononucleotide substrates demonstrate the accommodation of the nucleophilic 2-OH group in a binding pocket comprising the catalytic base (glutamate or histidine) and a charged hydrogen bond donor (lysine or histidine). Ab initio quantum chemical calculations performed on such Michaelis complexes of the mammalian RNase A (EC 3.1.27.5) and the microbial RNase T 1 (EC 3.1.27.3) show negative charge build up on the 2-oxygen upon substrate binding. The increased nucleophilicity results from stronger hydrogen bonding to the catalytic base, which is mediated by a hydrogen bond from the charged donor. This hitherto unrecognized catalytic dyad in ribonucleases constitutes a general mechanism for nucleophile activation in both enzymic and RNAcatalyzed phosphoryl transfer reactions.RNases have been the subject of landmark research in areas ranging from basic protein chemistry to enzymology to protein folding and crystallography. These enzymes catalyze the intramolecular nucleophilic displacement of the 5Ј-leaving group by the attacking 2Ј-hydroxyl group in RNA, forming a 2Ј,3Ј-cyclophosphate (see Fig. 1). The nucleophilic attack occurs inline, in a postulated trigonal bipyramidal transition state, implying a catalytic base and acid on either side of the scissile bond (1, 2). X-ray crystallographical and site-directed mutagenesis experiments have provided substantial insight in the structure-function relationship of two unrelated families of ribonucleases. RNase A, (bovine pancreatic ribonuclease A) (EC 3.1.27.5) (3) and RNase T 1 , (EC 3.1.27.3) (4, 5) (and references therein) are the best characterized members of the mammalian and the microbial enzymes, respectively. The mammalian enzymes depend on two histidines for acid/base catalysis, whereas a histidine/glutamic acid pair is found in the microbial enzymes (6).The active site of RNase T 1 is composed of the side chains of Tyr-38, His-40, Glu-58, Arg-77, His-92, and Phe-100 (see Fig. 2a). With the exception of Arg-77, all these amino acids have been shown to take part in catalysis (5). Both the His-40 and Glu-58 side chains are in the direct vicinity of the 2Ј-nucleophile. pH dependence studies have shown Glu-58 to be unprotonated and His-40 to be protonated at the onset of catalysis, proving that Glu-58 is the catalytic base accepting a proton from the 2Ј-nucleophile (7). The protonated His-92 located at the opposite side of the active site functions as the catalytic acid. Removal of the His-40 side chain leads to a 6500-fold decrease in the second-order rate constant k cat /K m suggesting an important catalytic role for this residue in the wild type enzyme. His-40 is believed to polarize the 2Ј-hydroxyl group and to properly orientate Glu-58 toward the substrate (8, 9). However, the actual role of this residue is not totally clarified at present. Despite the l...

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Geometry of the First Step in the Action of Ribonuclease-A

Cited by 146 publications

References 17 publications

Site-specific DNA transesterification catalyzed by a restriction enzyme

Site-specific DNA transesterification catalyzed by a restriction enzyme

Crystal structure of RNase A complexed with d(pA)4

A Nucleophile Activation Dyad in Ribonucleases

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