A re-investigation of the thio effect at the hammerhead cleavage site

Scott, Edmund C.; Uhlenbeck, Olke C.

doi:10.1093/nar/27.2.479

Cited by 85 publications

(60 citation statements)

References 34 publications

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“…4B-D). In contrast, the hammerhead ribozyme, which previously had been shown to exhibit a thio effect (e.g., see Scott and Uhlenbeck 1999), generates only 50% yield with Mg 2+ compared with Mn 2+ (data not shown). Therefore, the MR2, MR4, and MR11 ribozymes do not make use of metal-mediated ␤ catalysis, which is consistent with our hypothesis that these enzymes might be maximally using only ␣ and ␥ catalytic strategies.…”

Section: No Thio Effect For Candidate ␣␥ Enzymesmentioning

confidence: 78%

A common speed limit for RNA-cleaving ribozymes and deoxyribozymes

Breaker¹,

Emilsson²,

Lazarev³

et al. 2003

RNA

126

128

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It is widely believed that the reason proteins dominate biological catalysis is because polypeptides have greater chemical complexity compared with nucleic acids, and thus should have greater enzymatic power. Consistent with this hypothesis is the fact that protein enzymes typically exhibit chemical rate enhancements that are far more substantial than those achieved by natural and engineered ribozymes. To investigate the true catalytic power of nucleic acids, we determined the kinetic characteristics of 14 classes of engineered ribozymes and deoxyribozymes that accelerate RNA cleavage by internal phosphoester transfer. Half approach a maximum rate constant of ∼1 min −1 , whereas ribonuclease A catalyzes the same reaction ∼80,000-fold faster. Additional biochemical analyses indicate that this commonly encountered ribozyme "speed limit" coincides with the theoretical maximum rate enhancement for an enzyme that uses only two specific catalytic strategies. These results indicate that ribozymes using additional catalytic strategies could be made that promote RNA cleavage with rate enhancements that equal those of proteins.

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Section: No Thio Effect For Candidate ␣␥ Enzymesmentioning

confidence: 78%

A common speed limit for RNA-cleaving ribozymes and deoxyribozymes

Breaker¹,

Emilsson²,

Lazarev³

et al. 2003

RNA

126

128

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“…Also here, phosphorothioate studies helped to prove the coordination of divalent metal ions during catalysis [271,272]. resonance, respectively [274][275][276][277].…”

Section: Cadmium(ii)-rescue Experimentsmentioning

confidence: 94%

Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids

Sigel

Skilandat

Sigel

et al. 2012

Metal Ions in Life Sciences

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Cadmium(II), commonly classified as a relatively soft metal ion, prefers indeed aromaticnitrogen sites (e.g., N7 of purines) over oxygen sites (like sugar-hydroxyl groups). However, matters are not that simple, though it is true that the affinity of Cd(2+) towards ribose-hydroxyl groups is very small; yet, a correct orientation brought about by a suitable primary binding site and a reduced solvent polarity, as it is expected to occur in a folded nucleic acid, may facilitate metal ion-hydroxyl group binding very effectively. Cd(2+) prefers the guanine(N7) over the adenine(N7), mainly because of the steric hindrance of the (C6)NH(2) group in the adenine residue. This Cd(2+)-(N7) interaction in a guanine moiety leads to a significant acidification of the (N1)H meaning that the deprotonation reaction occurs now in the physiological pH range. N3 of the cytosine residue, together with the neighboring (C2)O, is also a remarkable Cd(2+) binding site, though replacement of (C2)O by (C2)S enhances the affinity towards Cd(2+) dramatically, giving in addition rise to the deprotonation of the (C4)NH(2) group. The phosphodiester bridge is only a weak binding site but the affinity increases further from the mono-to the di-and the triphosphate. The same also holds for the corresponding nucleotides. Complex stability of the pyrimidine-nucleotides is solely determined by the coordination tendency of the phosphate group(s), whereas in the case of purine-nucleotides macrochelate formation takes place by the interaction of the phosphate-coordinated Cd(2+) with N7. The extents of the formation degrees of these chelates are summarized and the effect of a non-bridging sulfur atom in a thiophosphate group (versus a normal phosphate group) is considered. Mixed ligand complexes containing a nucleotide and a further mono-or bidentate ligand are covered and it is concluded that in these species N7 is released from the coordination sphere of Cd(2+). In the case that the other ligand contains an aromatic residue (e.g., 2,2'-bipyridine or the indole ring of tryptophanate) intramolecular stack formation takes place. With buffers like Tris or Bistris mixed ligand complexes are formed. Cd(2+) coordination to dinucleotides and to dinucleoside monophosphates provides some insights regarding the interaction between Cd(2+) and nucleic acids. Cd(2+) binding to oligonucleotides follows the principles of coordination to its units. The available crystal studies reveal that N7 of purines is the prominent binding site followed by phosphate oxygens and other heteroatoms in nucleic acids. Due to its high thiophilicity, Cd(2+) is regularly used in so-called thiorescue experiments, which lead to the identification of a direct involvement of divalent metal ions in ribozyme catalysis.

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“…77 If the divalent metal ion binding at a certain position is required for catalysis, a large decrease in the reaction rate should be seen upon thio substitution at this position. However, the interpretation of thio affect results are sometimes inconclusive 21,23,78,79 especially when possible conformational changes induced by the sulfur are considered. Molecular simulation of thio-substituted phosphates and phosphoranes and their role on structure would aid in the interpretation of experimental thio effects.…”

Section: Thio Substitutionsmentioning

confidence: 99%

“…The product of the transesterification is a 5′-OH terminus and a 2′,3′-cyclic phosphate. Additional information about this mechanism has been obtained via kinetic isotope studies [18][19][20] and chemical modifications such as thio substitution [21][22][23] at the scissile phosphate, although the mechanistic interpretation of these experimental studies remains a topic of discussion and some debate. Time-resolved x-ray crystallography 10,24,25 has become a powerful tool to elucidate structural information at different stages along the catalytic reaction coordinate that provides valuable insight into ribozyme activity.…”

Section: Introductionmentioning

confidence: 99%

CHARMM force field parameters for simulation of reactive intermediates in native and thio‐substituted ribozymes

et al. 2006

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Force field parameters specifically optimized for residues important in the study of RNA catalysis are derived from density-functional calculations in a fashion consistent with the CHARMM27 allatom empirical force field. Parameters are presented for residues that model reactive RNA intermediates and transition state analogs, thio-substituted phosphates and phosphoranes, and bound Mg 2+ and di-metal bridge complexes. Target data was generated via density-functional calculations at the B3LYP/6-311++G(3df,2p)//B3LYP/6-31++G(d,p) level. Partial atomic charges were initially derived from the CHelpG electrostatic potential fitting and subsequently adjusted to be consistent with the CHARMM27 charges and Lennard-Jones parameters were determined to reproduce interaction energies with water molecules. Bond, angle and torsion parameters were derived from the density-functional calculations and renormalized to maintain compatibility with the existing CHARMM27 parameters for standard residues. The extension of the CHARMM27 force field parameters for the non-standard biological residues presented here will have considerable use in simulations of ribozymes, including the study of freeze-trapped catalytic intermediates, metal ion binding and occupation, and thio effects.

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A re-investigation of the thio effect at the hammerhead cleavage site

Cited by 85 publications

References 34 publications

A common speed limit for RNA-cleaving ribozymes and deoxyribozymes

A common speed limit for RNA-cleaving ribozymes and deoxyribozymes

Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids

CHARMM force field parameters for simulation of reactive intermediates in native and thio‐substituted ribozymes

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