Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

Abstract

Compared to protein enzymes, our knowledge about how RNA accelerates chemical reactions is rather limited. The crystal structures of a ribozyme that catalyzes Diels–Alder reactions suggest a rich tertiary architecture responsible for catalysis. In this study, we systematically probe the relevance of crystallographically observed ground-state interactions for catalytic function using atomic mutagenesis in combination with various analytical techniques. The largest energetic contribution apparently arises from the precise shape complementarity between transition state and catalytic pocket: A single point mutant that folds correctly into the tertiary structure but lacks one H-bond that normally stabilizes the pocket is completely inactive. In the rate-limiting chemical step, the dienophile is furthermore activated by two weak H-bonds that contribute ∼7–8 kJ/mol to transition state stabilization, as indicated by the 25-fold slower reaction rates of deletion mutants. These H-bonds are also responsible for the tight binding of the Diels–Alder product by the ribozyme that causes product inhibition. For high catalytic activity, the ribozyme requires a fine-tuned balance between rigidity and flexibility that is determined by the combined action of one inter-strand H-bond and one magnesium ion. A sharp 360° turn reminiscent of the T-loop motif observed in tRNA is found to be important for catalytic function.

A Selective Na+ Aptamer Dissected by Sensitized Tb3+ Luminescence

“…Because A10C showed good binding to Na + , the N7 site is unlikely to directly coordinate Na + , but it might form a hydrogen bond with other nucleotides for structural stabilization. For example, in a Diels–Alderase ribozyme, the A18 hydrogen bond with U8 to form a reverse Hoogsteen pair through N7 and exocyclic amine groups, which is important for its activity . Further studies are needed to fully understand the role of A10; this experiment has pointed out some directions.…”

Nucleic Acid Catalysis: Metals, Nucleobases, and Other Cofactors

“…163 This hydrogen bond is an important interstrand interaction that stabilizes the ribozyme and forms the “roof” of the catalytic pocket. Replacing the carbonyl O2 of U17 with an exocyclic amine completely abolishes ribozyme activity.…”

Preorganized Hydrogen Bond Donor Catalysts: Acidities and Reactivities

“…This enabled 3(2) and Schreiner's thiourea ((3,5-(CF 3 ) 2 C 6 H 3 NH) 2 CS, 6) 15 to be examined, but since CD 3 CN is a hydrogen bond acceptor its presence was expected to slow down the reaction. This was observed for 3 (3) in that the presence of CD 3 CN was found to retard the transformation by a factor of ∼4 (entries 2 and 6, Table 2). Nevertheless, both 3(2) and 3(3) were found to be more effective catalysts than 6 despite the thiourea being more acidic than 3(2) in DMSO (i.e., pK a = 8.5 (6) and 9.5 (3(2))).…”

“…All of the calculations carried out in this work were performed at the Minnesota Supercomputer Institute for Advanced Computational Research using Gaussian 09. 49 Full geometry optimizations and vibrational frequencies were carried out on triols 3(0)−3 (3) and their conjugate bases with the B3LYP density functional and the 6-31+G(d,p) basis set. 38,39 The most stable conformers located were reoptimized with the larger 6-311+G(d,p) basis set as well as with the M06-2X functional and the aug-cc-pVDZ basis set.…”