Ribozyme active site

Figure 6.17 Talo-5 C-methyl substituent at the hammerhead ribozyme active site.

Evidence for Acid/Base Catalysis in the Hepatitis Delta Virus Ribozyme Active Site... 

Figure 5 Proposed acid/base catalytic interactions in the HDV ribozyme active site. The cleavage site for the HDV ribozyme is shown with the nucleotide 5 to the site of bond cleavage shown in red and the nucleotide 3 to that site shown in blue. Two proposed mechanisms for the function of the C76 cytisine nucleobase and a hydrated active site metal ion are shown in which C76 acts as either an acid (left) or base (right) as described in the text.

Figure 6 Proposed divalent metal ion coordination interactions involved in metal ion catalysis by the GI intron ribozyme active site. The coordination interactions determined by substrate PS and 2 amino modification and quantitative metal rescue are depicted on the left. The individual interacting functional groups are shown in red. The observed metal ions (green spheres) in the crystal structure of the GI ribozyme active site is shown on the right and the metal ligands identified by functional studies are shown as red spheres. Adapted from Reference 56.

An RNA Diels-Alderase ribozyme recently developed that catalyses the formation of carbon-carbon bonds between a tethered anthracene diene and a biotinylated maleimide dienophile. The ribozyme active site has been further characterised by chemical substitution of the diene and dienophile. It was shown that the diene must be an anthracene, and substitution only at specific sites is permitted. The dienophile must be a maleimide with an unsubstituted double bond. The RNA-diene interaction was found to be governed preferentially by stacking interactions. A ribozyme has been selected that catalyses the synthesis of dipeptides using an aminoacyl-adenylate substrate. The ribozyme catalysed the formation of 30 different dipeptides, many with rates similar to that of the Met-Phe dipeptide used in the selection process. 

Figure 1 Schematic view of the coordination sites in the hammerhead ribozyme active site. Upper left The coordination pattern of Mg2 in the C-site coordinated to G10.1 N7 and k9 Ow. Upper right The coordination pattern of Mg2 in the B-site bridging A9 02p and Cl.LO of the scissile phosphate. Lower Coordination sites for Na in the hammerhead ribozyme active site found in the RT-Na and dRT-Na simulations. Red numbers next to the coordination sites are the scores used to calculate the coordination index (see text). M, involves direct binding to A9 02P and C.1 02P and indirect binding to G10.1 N 7 through a water molecule. M2 involves direct binding to CT7 02- and C.l 02p M3 involves direct binding to CI7 02 and is positioned toward the outside of the active site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this book.)...

Fig. 5-2 The ribozyme active site is formed when the RNA folds into a hairpin structure. The cleaved (scissile) phosphate-ester bond is flanked by a guanine (Gg) and an adenine (Agg) moiety that stabilize a transition state by hydrogen bonding. The structure shown was prepared from the coordinates of the crystal structure of a vanadium salt, hence the V in the center of the structure. The dashed lines denote hydrogen bonds.

The a-amino group of the new aminoacyl-tRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA occupying the P site (peptidyl or polypeptide site). At initiation, this site is occupied by aminoacyl-tRNA mef. This reaction is catalyzed by a peptidyltransferase, a component of the 285 RNA of the 605 ribosomal subunit. This is another example of ribozyme activity and indicates an important—and previously unsuspected—direct role for RNA in protein synthesis (Table 38-3). Because the amino acid on the aminoacyl-tRNA is already activated, no further energy source is required for this reaction. The reaction results in attachment of the growing peptide chain to the tRNA in the A site. 

Figure 14-7. Snapshots of the active site structures near the transition state of (top) the nucleophilic attack and (bottom) the exocyclic cleavage for the in-line monoanionic O2p mechanism of cleavage transesterification in the hairpin ribozyme. The yellow and red colored cartoon is for the substrate and ribozyme strands, respectively, and water molecules interacting with non-bridging oxygens and O5/ are shown...

Figure 14-8. The 3D density contour maps (yellow) of Na+ ion distributions derived from the activated precursor simulation. The hammerhead ribozyme is shown in blue with the active site in red. Only the high-density contour is shown here to indicate the electrostatic recruiting pocket formed in the active site...

However, there are a number of other miscellaneous biological roles played by this complex. The [Co(NH3)6]3+ ion has been shown to inhibit the hammerhead ribozyme by displacing a Mn2+ ion from the active site.576 However, [Co(NH3)6]3+ does not inhibit ribonuclease H (RNase),577 topoisomerase I,578 or hairpin ribozyme,579 which require activation by Mg2+ ions. The conclusions from these studies were that an outer sphere complex formation between the enzyme and Mgaq2+ is occuring rather than specific coordination of the divalent ion to the protein. These results are in contrast to DNase I inhibition by the same hexaammine complex. Inhibition of glucose-induced insulin secretion from pancreatic cells by [Co(NH3)6]3+ has been found.580 Intracellular injection of [Co(NH3)6]3+ into a neurone has been found to cause characteristic changes to the structure of its mitochondria, and this offers a simple technique to label neuronal profiles for examination of their ultrastructures.581... 

The peptidyl transferase centre of the ribosome is located in the 50S subunit, in a protein-free environment (there is no protein within 15 A of the active site), supporting biochemical evidence that the ribosomal RNA, rather than the ribosomal proteins, plays a key role in the catalysis of peptide bond formation. This confirms that the ribosome is the largest known RNA catalyst (ribozyme) and, to date, the only one with synthetic activity. Adjacent to the peptidyl transferase centre is the entrance to the protein exit tunnel, through which the growing polypeptide chain moves out of the ribosome. 

The intron group I ribozymes feature common secondary structure and reaction pathways. Active sites capable of catalyzing consecutive phosphodi-ester reactions produce properly spliced and circular RNAs. Ribozymes fold into a globular conformation and have solvent-inaccessible cores as quantified by Fe(II)-EDTA-induced free-radical cleavage experiments. The Tetrahy-mem group I intron ribozyme catalyzes phosphoryl transfer between guanosine and a substrate RNA strand—the exon. This ribozyme also has been proposed to use metal ions to assist in proper folding, to activate the nucleophile, and to stabilize the transition state. ... 

In the following year, Scott and co-workers solved the X-ray crystallographic structure of an all-RNA hammerhead ribozyme with a 2 -OCH3 group incorporated at the active site cytosine (Cn) to prevent cleavage (PDB IMME). This structure differed from that of IHMH in several important ways (1) it was an all-RNA ribozyme rather than an RNA-DNA hybrid (2) the connectivity of the ribozyme backbone strands was different (for instance... 

Another naturally occurring ribozyme which catalyzes phosphodiester transfer reactions is the hairpin ribozyme. The hairpin ribozyme has been the subject of a number of excellent review articles [24,25]. Several independent studies performed recently have indicated that the hairpin ribozyme has an interesting feature which distinguishes it from the aforementioned ribozymes mechanistically While the HHR, the group I intron, the HDV ribozyme and many other ribozymes that we are going to meet in this review are metalloenzymes and require divalent metal ions in their active sites for functional group activation, divalent metals ions only play a passive role (they are mainly required for cor-... 

In our recent study of reaction kinetics, we observed an unusual phenomenon when we analyzed the activity of a hammerhead ribozyme as a function of the concentration of Na ions on a background of a low concentration of either Mn or Mg ions [82]. At lower concentrations of Na ions, Na ions had an inhibitory effect on ribozyme activity, whereas at higher concentrations, Na ions had a rescue effect. We propose that these observations can be explained if we accept the existence of two kinds of metal-binding site that have different affinities. Our data also support the two-phase folding theory [77-80, Fig. 7], in which divalent metal ions in the ri-bozyme-substrate complex have lower and higher affinities, as proposed by Lilley and coworkers on the basis of their observations of ribozyme complexes in the ground state. 

This inhibitory effect of NaCl can be explained on the basis of the data from Horton et al. [81] that is described above. The Na ions remove Mn ions from the lower affinity site(s), which is somehow involved in the ribozyme activity, from the ribozyme-substrate complex. When Hammann et al. used ions in their NMR analysis, they noticed that the apparent Ka for the lower affinity ions depended on the concentration of Na ions [80]. An increase in the background concentration of NaCl from 10 mmol/1 to 50 mmol/1 weakened the affinity of the Mg ions for the complex. Their observations also reconcile with the observed inhibition by Na ions in our study, with the competitive removal of Mg /Mn ions from the ribozyme-substrate complex. 

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