Chemical Reactions with RNA and DNA Enzymes

Institut filr Pharmazie und Molekulare Biotechnologie (IPMB), Universitat Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany 

As enzymes encapsulate multiple functionalities within their catalytic cavity, they have also served as a major source of inspiration for the fields of biomimetic chemistry and supramolecular catalysis. Early mechanistic theories about how enzymes work have prompted scientists from various fields to explore similar approaches for synthetic systems. One of these approaches is host-guest catalysis, where one or more substrates are bound in a cavity next to the catalytically active site. 

Enzymes are, however, much more than just a combination of substrate binding site and the catalytically active site. Important contributions to enzymatic catalysis arise from substrate preorganization, restriction of substrate motion, catalyst dynamics, transition state binding, and desolvation of the substrates, and natural evolution has used these 

Molecular Encapsidation Organic Reactions in Constrained Systems 2010 John Wiley Sons, Ltd 

While enzymological research over several decades has provided a rather detailed understanding of how certain enzymes accelerate chemical reactions, knowledge about ribozyme mechanisms and catalytic strategies is scarce in comparison. For some natural ribozymes, mechanistic and structural studies were carried out, but artificial ribozymes remain largely uncharacterized. Therefore it is unclear in many cases whether these catalysts are passive containers working solely by substrate pre-organization, or whether there are specific effects on the activation parameters of the reaction. 

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DNA polymerase I is a nonessential enzyme, since viable E. coli mutants lack it (pol A). This conclusion is complicated, however, since the enzyme catalyzes three separate chemical reactions. It polymerizes deoxyribonucleoside triphosphates, and it has two exonucleolytic activities, a 3 to 5 activity and a 5 to 3 activity. The pol A - mutants lack only the polymerization activity. Other mutants lacking both the polymerase and the 5 to 3 exonuclease activity are lethal. Thus the exonuclease function is the more important one. This fits with the role of this enzyme in removing damaged DNA segments (DNA repair) and in removing covalently attached RNA from DNA chains. We will later see that small RNAs serve as primers of DNA synthesis. 

The discovery of self-splicing introns showed that RNA could catalyse chemical reactions. Yet, unlike proteins, RNA has no functional groups with pKa values and chemical properties similar to those considered to be important in protein-based enzymes. Steitz and Steitz (1993) postulated that two metal ions were essential for catalysis by ribozymes using a mechanism similar to DNA cleavage, in which a free 3 OH is produced. They proposed,... 

These footprinting analyses, based on enzymic and chemical digestion, are now widely used to define DNA (and RNA) and their complexes with various ligands. Recently active radical probes have been used as footprinting agents in protection assays in a variety of systems (e.g., Tullius and Dombroski, 1986 Chalepakis and Beato, 1989 Hayes and Tullius, 1989 Schickor et al., 1990). Such probes rely on active radical intermediates, most likely hydroxyl radicals, released by Fe(II) in the presence of an electron donor, probably via a Fenton reaction. In addition, hydroxyl radicals also appear to react with DNA in a conformation-specific manner which may allow some prediction of DNA secondary structure (see Burkhoft and Tullius, 1987 Zorbas et al., 1989 Lu et al., 1990). 

The DNA and RNA polymerase reactions, as well as the reverse transcriptase and polynucleotide phosphorylase reactions, proceed with inversion of configuration at Pa of the nucleoside triphosphate (45-50). Thus, an uneven number of displacements at phosphoms is involved in the chemical reaction mechanism, and the stereochemistry provides no evidence for the involvement of a covalent nucleotidyl-enzyme as an intermediate on the catalytic pathway. No other evidence for such an intermediate is available. Therefore, it must be concluded that the physicochemical requirements for nucleotidyl group transfer, substrate recognition, and movement along the template are derived fiom binding interactions between the enzyme and its template and substrate rather than through nucleophilic catalysis. This is also true of polynucleotide phosphorylase and other nucleotidyltransferases that catalyze reactions of polynucleotides (51, 52). 

Polymerase enzymes are used extensively in the laboratory for the synthesis and manipulation of DNA and RNA (44). One of the most important applications of enzymatic DNA synthesis is in the polymerase chain reaction (PGR), which is used to amplify minute quantities of DNA from a variety of soin-ces, inclnd-ing crime scene evidence, archaeological specimens, or medical samples (45). In PGR, two short oligonucleotide primers are first synthesized by chemical methods. These primers are complementary to the 5 -ends of the two strands which form the DNA duplex of interest (Fig. 11). A large excess of the primers is mixed with the target DNA along with a DNA polymerase enzyme and the nucleoside triphosphate monomers. The sample is heated to disrupt the target duplex. Then, as the... 

Although the list of transmethylation reactions in which S-adenosylmethionine can function as the methyl donor could now undoubtedly be greatly expanded, the more recently added reactions, with the exception of the carbon methylations discussed below (Section II,C,6), appear from the chemical point of view to be further examples of types of reactions which were already known. Therefore, no complete compilation will be attempted. From the physiological standpoint, on the other hand, some of the reactions recently studied may be of great interest. For example, systems have now been reported for the enzymic methylation of RNA (Srinavasan and Borek, 1964 Rodeh et al, 1967), DNA (Oda and Marmur, 1966 Kalousek and Morris, 1969), pectin (Kauss and Hassid, 1967), and protein (Comb et al, 1966 Paik and Kim, 1968 Liss et al, 1969). The specific modifications of the properties of these biopolymers consequent to methylation may, of course, be important in... 

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