Limits...
'In-line attack' conformational effect plays a modest role in an enzyme-catalyzed RNA cleavage: a free energy simulation study.

Min D, Xue S, Li H, Yang W - Nucleic Acids Res. (2007)

Bottom Line: Since the proposal of 'in-line attack' conformation as a possibly important intermediate in RNA cleavage, its structure has been captured in various protein and RNA enzymes; these structures strengthen the belief that this conformation plays an essential role in the catalysis of RNA cleavage.As generally discussed, this intermediate structure can be involved in energy barrier reduction in two possible ways, e.g. through either conformational effect or electrostatic effect.In order to quantitatively elucidate the contribution of conformational effect in this type of enzyme catalysis, free energy simulations were performed on the RNA structures both in a splicing endonuclease complex and in the aqueous solution.

View Article: PubMed Central - PubMed

Affiliation: School of Computational Science, Florida State University, Tallahassee, FL 32306, USA.

ABSTRACT
Since the proposal of 'in-line attack' conformation as a possibly important intermediate in RNA cleavage, its structure has been captured in various protein and RNA enzymes; these structures strengthen the belief that this conformation plays an essential role in the catalysis of RNA cleavage. As generally discussed, this intermediate structure can be involved in energy barrier reduction in two possible ways, e.g. through either conformational effect or electrostatic effect. In order to quantitatively elucidate the contribution of conformational effect in this type of enzyme catalysis, free energy simulations were performed on the RNA structures both in a splicing endonuclease complex and in the aqueous solution. Our free energy simulation results revealed that the 'in-line attack' conformational effect plays a modest role in facilitating the reaction rate enhancement (approximately 12-fold) compared with the overall 10(12)-fold rate increase. The close agreement between the present computational estimation and an experimental measurement on the spontaneous RNA cleavage in an in vitro evolved ATP aptamer motives us to realize that the conformation distribution of an enzyme substrate prior to rather than after its binding determines the upper bound of the rate enhancement ability through the conformational strategy.

Show MeSH
‘In-line attack’ conformation of a pre-cleaved RNA substrate in splicing endonuclease (A) comparison of a regular RNA conformation (upper) and an ‘in-line attack’ conformation (lower) in term of dihedral angle Φ(C3′–O3′–P–O5′); (B) illustration of ‘in-line attack’ conformer of RNA substrate inside enzyme (gray surface) binding pocket.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1919504&req=5

Figure 2: ‘In-line attack’ conformation of a pre-cleaved RNA substrate in splicing endonuclease (A) comparison of a regular RNA conformation (upper) and an ‘in-line attack’ conformation (lower) in term of dihedral angle Φ(C3′–O3′–P–O5′); (B) illustration of ‘in-line attack’ conformer of RNA substrate inside enzyme (gray surface) binding pocket.

Mentions: RNA cleaving enzymes can bring up to 1012-fold rate enhancement, as compared with the uncatalyzed reaction in the aqueous solution (2). This large rate acceleration can be attributed to various factors such as conformational effect and electrostatic transition state stabilization effect (7). Recently, conformational effect has been invoked to understand catalytic strategies in various enzymes (8–11), such as in chorismate mutase, where this effect alone can contribute up to 103-fold rate enhancement (12). Upon the substrate binding, a tight conformation, which is structurally close to the corresponding transition state, is usually formed within the bound substrate prior to any chemical steps. Specifically, in the RNA intra-molecular phosphoester transfer reactions, such tight conformation is referred as an ‘in-line attack’ conformation (13,14). Here, the attacking atom O2′ is placed opposite to the leaving O5′ atom and aligned in the direction of the broken P–O5′ bond (Scheme 1B). This ‘in-line attack’ conformation has been observed in the structures of a hammerhead ribozyme (6) and two RNA cleaving enzymes bound to their RNA substrates (15,16) (Figure 1B). These structural data are consistent with the previous speculation that ‘in-line attack’ conformation can be important for RNA cleavage (14). In the present work, we wish to quantitatively evaluate how much the conformational effect of this ‘in-line attack’ structure contributes to the rate acceleration in an enzyme-catalyzed RNA cleavage reaction.Figure 1.


'In-line attack' conformational effect plays a modest role in an enzyme-catalyzed RNA cleavage: a free energy simulation study.

Min D, Xue S, Li H, Yang W - Nucleic Acids Res. (2007)

‘In-line attack’ conformation of a pre-cleaved RNA substrate in splicing endonuclease (A) comparison of a regular RNA conformation (upper) and an ‘in-line attack’ conformation (lower) in term of dihedral angle Φ(C3′–O3′–P–O5′); (B) illustration of ‘in-line attack’ conformer of RNA substrate inside enzyme (gray surface) binding pocket.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919504&req=5

Figure 2: ‘In-line attack’ conformation of a pre-cleaved RNA substrate in splicing endonuclease (A) comparison of a regular RNA conformation (upper) and an ‘in-line attack’ conformation (lower) in term of dihedral angle Φ(C3′–O3′–P–O5′); (B) illustration of ‘in-line attack’ conformer of RNA substrate inside enzyme (gray surface) binding pocket.
Mentions: RNA cleaving enzymes can bring up to 1012-fold rate enhancement, as compared with the uncatalyzed reaction in the aqueous solution (2). This large rate acceleration can be attributed to various factors such as conformational effect and electrostatic transition state stabilization effect (7). Recently, conformational effect has been invoked to understand catalytic strategies in various enzymes (8–11), such as in chorismate mutase, where this effect alone can contribute up to 103-fold rate enhancement (12). Upon the substrate binding, a tight conformation, which is structurally close to the corresponding transition state, is usually formed within the bound substrate prior to any chemical steps. Specifically, in the RNA intra-molecular phosphoester transfer reactions, such tight conformation is referred as an ‘in-line attack’ conformation (13,14). Here, the attacking atom O2′ is placed opposite to the leaving O5′ atom and aligned in the direction of the broken P–O5′ bond (Scheme 1B). This ‘in-line attack’ conformation has been observed in the structures of a hammerhead ribozyme (6) and two RNA cleaving enzymes bound to their RNA substrates (15,16) (Figure 1B). These structural data are consistent with the previous speculation that ‘in-line attack’ conformation can be important for RNA cleavage (14). In the present work, we wish to quantitatively evaluate how much the conformational effect of this ‘in-line attack’ structure contributes to the rate acceleration in an enzyme-catalyzed RNA cleavage reaction.Figure 1.

Bottom Line: Since the proposal of 'in-line attack' conformation as a possibly important intermediate in RNA cleavage, its structure has been captured in various protein and RNA enzymes; these structures strengthen the belief that this conformation plays an essential role in the catalysis of RNA cleavage.As generally discussed, this intermediate structure can be involved in energy barrier reduction in two possible ways, e.g. through either conformational effect or electrostatic effect.In order to quantitatively elucidate the contribution of conformational effect in this type of enzyme catalysis, free energy simulations were performed on the RNA structures both in a splicing endonuclease complex and in the aqueous solution.

View Article: PubMed Central - PubMed

Affiliation: School of Computational Science, Florida State University, Tallahassee, FL 32306, USA.

ABSTRACT
Since the proposal of 'in-line attack' conformation as a possibly important intermediate in RNA cleavage, its structure has been captured in various protein and RNA enzymes; these structures strengthen the belief that this conformation plays an essential role in the catalysis of RNA cleavage. As generally discussed, this intermediate structure can be involved in energy barrier reduction in two possible ways, e.g. through either conformational effect or electrostatic effect. In order to quantitatively elucidate the contribution of conformational effect in this type of enzyme catalysis, free energy simulations were performed on the RNA structures both in a splicing endonuclease complex and in the aqueous solution. Our free energy simulation results revealed that the 'in-line attack' conformational effect plays a modest role in facilitating the reaction rate enhancement (approximately 12-fold) compared with the overall 10(12)-fold rate increase. The close agreement between the present computational estimation and an experimental measurement on the spontaneous RNA cleavage in an in vitro evolved ATP aptamer motives us to realize that the conformation distribution of an enzyme substrate prior to rather than after its binding determines the upper bound of the rate enhancement ability through the conformational strategy.

Show MeSH