Direct Raman Measurement of an Elevated Base p<i>K</i><sub>a</sub> in the Active Site of a Small Ribozyme in a Precatalytic Conformation

Guo, Min; Spitale, Robert C.; Volpini, Rosaria; Krucinska, J.; Cristalli, Gloria; Carey, Paul R.; Wedekind, Joseph E.

doi:10.1021/ja9060883

Cited by 49 publications

(67 citation statements)

References 26 publications

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“…Hence, the lack of an effective base and an effective acid and an increase of the energy of the transition state are possibly responsible for the sixth order of magnitude decrease in the cleavage rate of the G40A mutant in the presence of GlcN6P. Therefore, our results suggest that the general acid (the amine group of GlcN6P) has a pK a of about 6 and the general base (if G is the base) has a pK a of about 9.2, similar to the possibility in the hairpin ribozyme, with pK a s of 5.4 (A38; the acid) and 9.5 (Guo et al 2009; Ferre-D'Amare 2010), respectively.…”

Section: Resultssupporting

confidence: 66%

“…2;Hampel and Tinsley 2006;Klein and Ferre-D'Amare 2006;Cochrane et al 2007Cochrane et al , 2009). The general acid-base catalytic mechanism has been extensively studied (Thompson and Raines 1994;Breslow and Chapman 1996;Sowa et al 1997;Bevilacqua 2003;Emilsson et al 2003;Bevilacqua et al 2004;Lonnberg and Lonnberg 2005;Bevilacqua and Yajima 2006) and is common in both protein (Thompson and Raines 1994;Breslow and Chapman 1996;Sowa et al 1997) and nucleic acid chemistry (Nakano et al 2000;Han and Burke 2005;Guo et al 2009). In glmS, the proposed cleavage mechanism is similar to that of other self-cleaving ribozymes, in which cleavage is initiated by the binding of GlcN6P.…”

Section: Introductionmentioning

confidence: 99%

“…In HDV, the pK a of N3 of C76 is perturbed toward neutrality, and the perturbed C76 has been shown to act as the general acid in the self-cleavage, based on experimental and computational studies (Nakano et al 2000;Shih and Been 2001;Krasovska et al 2005). Also, using Raman crystallography, Guo et al (2009) showed that the pK a of N1 of A38 in the hairpin ribozyme (HPRZ) FIGURE 1. X-ray crystal structure of glmS ribozyme (A) from Thermoanaerobacter tengcongensis complexed with GlcN6P (B), shown in red.…”

Section: Introductionmentioning

confidence: 99%

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Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Yao¹,

Hamelberg²

2010

RNA

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The GlmS ribozyme is believed to exploit a general acid-base catalytic mechanism in the presence of glucosamine-6-phosphate (GlcN6P) to accelerate self-cleavage by approximately six orders of magnitude. The general acid and general base are not known, and the role of the GlcN6P cofactor is even less well understood. The amine group of GlcN6P has the ability to either accept or donate a proton and could therefore potentially act as an acid or a base. In order to decipher the role of GlcN6P in the self-cleavage of glmS, we have determined the preferred protonation state of the amine group in the wild-type and an inactive G40A mutant using molecular dynamics simulations and free energy calculations. Here we show that, upon binding of GlcN6P to wild-type glmS, the pK a of the amine moiety is altered by the active site environment, decreasing by about 2.2 from a solution pK a of about 8.2. On the other hand, we show that the pK a of the amine group slightly increases to about 8.4 upon binding to the G40A inactive mutant of glmS. These results suggest that GlcN6P acts as a general acid in the self-cleavage of glmS. Upon binding to glmS, GlcN6P can easily release a proton to the 59-oxygen of G1 during self-cleavage of the backbone phosphodiester bond. However, in the G40A inactive mutant of glmS, the results suggest that the ability of GlcN6P to easily release its proton is diminished, in addition to the possible lack of G40 as an effective base.

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Section: Resultssupporting

confidence: 66%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Yao¹,

Hamelberg²

2010

RNA

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“…Poisson-Boltzmann calculations (Tang et al 2007) and Raman spectroscopy (Guo et al 2009) have shown that A38 has an elevated pK a (pK a values of 5.9 and 5.46, respectively), consistent with the observed pH dependence of activity arising from proton transfer at this nucleobase. Exogenous base rescue and substitution with other nucleobases have demonstrated the importance of both the N1 and N6 of A38 for full activity (Kuzmin et al 2005).…”

Section: Does the Hairpin Ribozyme Share The Same Catalytic Mechanism?supporting

confidence: 57%

Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid–base catalysis? A critical assessment of available experimental data

Wilson

Lilley²

2010

RNA

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The active centers of the hairpin and VS ribozymes are both generated by the interaction of two internal loops, and both ribozymes use guanine and adenine nucleobases to accelerate cleavage and ligation reactions. The centers are topologically equivalent and the relative positioning of key elements the same. There is good evidence that the cleavage reaction of the VS ribozyme is catalyzed by the guanine (G638) acting as general base and the adenine (A756) as general acid. We now critically evaluate the experimental mechanistic evidence for the hairpin ribozyme. We conclude that all the available data are fully consistent with a major contribution to catalysis by general acid-base catalysis involving the adenine (A38) and guanine (G8). It appears that the two ribozymes are mechanistically equivalent.

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“…NMR with isotopically labeled RNAs and, more recently, Raman crystallography have been used to measure microscopic pK a values for protonation of N3 of pyrimidines and N1 of purines in catalytic RNAs (42,47,48,51). NMR analyses of A38 protonation in an isolated loop B domain of the Hp Rz gave a pK a value of 4.89 (40).…”

Section: Discussionmentioning

confidence: 99%

The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine

Cottrell

Scott

Fedor

2011

Journal of Biological Chemistry

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Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pK a values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK a for ionization of 8azaA at position 38 with the apparent pK a for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK a values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5-oxygen-phosphorus bond.Hairpin ribozymes (Hp Rz) 2 belong to one of several families of small self-cleaving RNAs that serve as useful models of RNA catalysis because they are relatively simple and amenable to chemogenetic analyses (1). The Hp Rz remains functional in the absence of divalent metals, relying exclusively on nucleotide functional groups for catalytic chemistry (2-5). High resolution structures of the Hp Rz bound to transition state mimics show two active site purines, G8 and A38, positioned in a manner similar to the two histidines in RNase A that mediate general acid-base catalysis of the same reaction ( Fig. 1A) (6 -8), leading to the proposal that the Hp Rz uses the same concerted general acid-base mechanism (Fig. 1B) (9). A38(N1) is near the 5Ј-oxygen of the reactive phosphodiester, and A38(N6) lies within hydrogen bonding distance of the pro-R P non-bridging oxygen. Exogenous nucleobase rescue experiments confirmed that the amidine group of A38 interacts with the transition state, but its precise role remained unclear (10). In a general acid-base model, the cationic protonated form of A38 would act as a general acid during cleavage to donate a proton to the 5Ј-oxygen leaving group as the 5Ј-oxygen-phosphorus bond breaks. During the reverse ligation reaction, unprotonated A38 would accept a proton to activate the nucleophilic 5Ј-oxygen for attack on phosphorus as the 5Ј-oxygen-phosphorus bond forms. Cleavage and ligation rate constants both increase with increasing pH and exhibit the same apparent pK a values near 6.5 (3), consistent with the law of microscopic reversibility (11). The pH dependence of reaction kinetics can provide information about the identity of general acid-base catalyst when an apparent pK a value clearly corresponds to the microscopic pK a value for a particular active site functional group. However, no RNA functional groups exhibit protonation equilibria near pH 6.5, at least as free nucleotides in solu...

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Direct Raman Measurement of an Elevated Base pK_a in the Active Site of a Small Ribozyme in a Precatalytic Conformation

Cited by 49 publications

References 26 publications

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid–base catalysis? A critical assessment of available experimental data

The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine

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Direct Raman Measurement of an Elevated Base pKa in the Active Site of a Small Ribozyme in a Precatalytic Conformation

Cited by 49 publications

References 26 publications

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid–base catalysis? A critical assessment of available experimental data

The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine

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Direct Raman Measurement of an Elevated Base pK_a in the Active Site of a Small Ribozyme in a Precatalytic Conformation