Phosphodiester bond hydrolysis,

Schulz, W. G. Nieman, R. A. Skibo, E. B. Evidence for DNA phosphate backbone alkylation and cleavage by pyrrolo[l,2-a] benzimidazoles, small molecules capable of causing sequence specific phosphodiester bond hydrolysis. Proc. Natl. Acad. Sci. USA 1995, 92, 11854-11858. 

The study of posttranscriptional processing of RNA molecules led to one of the most exciting discoveries in modern biochemistry—the existence of RNA enzymes. The best-characterized ribozymes are the self-splicing group I introns, RNase P, and the hammerhead ribozyme (discussed below). Most of the activities of these ribozymes are based on two fundamental reactions transesterification (Fig. 26-13) and phosphodiester bond hydrolysis (cleavage). The substrate for ribozymes is often an RNA molecule, and it may even be part of the ribozyme itself. When its substrate is RNA, an RNA cat-... 

Figure 6 (a) The catalytic mechanism of RNase k, including the postulated transition state, (b) Bait-and-switch hapten 6 elicited antibody MATT.F-1 that catalyzes phosphodiester bond hydrolysis of substrate 7. Transition state analog hapten 8 also elicited catalytic antibodies but with slower rates. 

Site-specific synthetic ribonucleases based on conjugates of oligonucleotides with metal-independent organic catalysts of phosphodiester bond hydrolysis 02IZV1025. 

Friedhoff, P. Franke, I. Krause, K L. Pingoud, A., Cleavage experiments with deoxythymidine 3 -5 -bis-(p-nitrophenyl phosphate) suggest that the homing endonuclease l-Ppol follows the same mechanism of phosphodiester bond hydrolysis as the non-specific Serratia nuclease. FEBS Lett 1999,443,209-214. 

Fig. 3.7. Differences between in-line and adjacent mechanism of phosphodiester bond hydrolysis, (a) First step (transesterification) in the RNase catalyzed hydrolysis of RNA illustrating two possible stereochemical pathways The in-line mechanism allows a direct displacement while the adjacent mechanism requires a pseudo-rotation, (b) Second step (hydrolysis) in the RNase catalyzed hydrolysis of RNA illustrating two possible stereochemical pathways. Again, the in-line mechanism allows a direct displacement to form the 3 -phosphate, while the adjacent mechanism requires a pseudorotation (26).

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

FIGURE 11.29 The vicinal—OH groups of RNA are susceptible to nucleophilic attack leading to hydrolysis of the phosphodiester bond and fracture of the polynucleotide chain DNA lacks a 2 -OH vicinal to its 3 -0-phosphodiester backbone. Alkaline hydrolysis of RNA results in the formation of a mixture of 2 - and 3 -nucleoside monophosphates. 

DNA is not susceptible to alkaline hydrolysis. On the other hand, RNA is alkali labile and is readily hydrolyzed by dilute sodium hydroxide. Cleavage is random in RNA, and the ultimate products are a mixture of nucleoside 2 - and 3 -monophosphates. These products provide a clue to the reaction mechanism (Figure 11.29). Abstraction of the 2 -OH hydrogen by hydroxyl anion leaves a 2 -0 that carries out a nucleophilic attack on the phosphorus atom of the phosphate moiety, resulting in cleavage of the 5 -phosphodiester bond and formation of a cyclic 2, 3 -phosphate. This cyclic 2, 3 -phosphodiester is unstable and decomposes randomly to either a 2 - or 3 -phosphate ester. DNA has no 2 -OH therefore DNA is alkali stable. 

Staphylococcal nuclease (SNase) is a single-peptide chain enzyme consisting of 149 amino acid residues. It catalyzes the hydrolysis of both DNA and RNA at the 5 position of the phosphodiester bond, yielding a free 5 -hydroxyl group and a 3 -phosphate monoester... 

The overall catalytic rate constant of SNase is (see, for example, Ref. 3) kcat — 95s 1 at T = 297K, corresponding to a total free energy barrier of Ag at = 14.9 kcal/mol. This should be compared to the pseudo-first-order rate constant for nonenzymatic hydrolysis of a phosphodiester bond (with a water molecule as the attacking nucleophile) which is 2 x 10 14 s corresponding to Ag = 36 kcal/mol. The rate increase accomplished by the enzyme is thus 101S-1016, which is quite impressive. 

While formation of a dinucleotide may be represented as the elimination of water between two monomers, the reaction in fact strongly favors phosphodiester hydrolysis. Phosphodiesterases rapidly catalyze the hydrolysis of phosphodiester bonds whose spontaneous hydrolysis is an extremely slow process. Consequently, DNA persists for considerable periods and has been detected even in fossils. RNAs are far less stable than DNA since the 2khydroxyl group of RNA... 

Figure 15 Hydrolysis of phosphodiester bonds catalyzed by antibody MATT.F-1 raised against hapten 19 according to the bait and switch strategy.

Figure 10.7 Molecular structure of a section of a single strand of DNA. Note that interatomic distances are considerably distorted in this representation. Bonds (i) and (ii) are positions of hydrolysis of the phosphodiester bond by different nucleases.

Dimethyl sulfate is an effective methylating agent (see Section 7.13.1). Methylation of the purine rings in gnanine and adenine makes them susceptible to hydrolysis and snbseqnent rnptnre. This, in tnm, makes the glycosidic bond vnlnerable to attack, and the heterocycle is displaced from the phosphodiester. The phosphodiester bond can then cleaved by basic hydrolysis (aqueons piperidine). 

DNA-(apurinic or apyrimidinic site) lyase [EC 4.2.99.18, formerly EC 3.1.25.2] acts on the C-Q-P bond 3 to the apurinic or apyrimidinic site in DNA. This bond is broken by a /3-elimination reaction, leaving a 3 -terminal unsaturated sugar and a product with a terminal 5 -phosphate. Note that this nicking of the phosphodiester bond is a lyase-type reaction, not hydrolysis. 

Deoxyribonucleases (DNAses) catalyze hydrolysis of phosphodiester bonds in the DNA backbone, leaving a 5 -phosphate and a 3 -hydroxyl on the ends. 

It is evident that most future work with irradiated polynucleotides will have to employ techniques such as these. Many pertinent observations were made about the effect of irradiation of the poly U upon enzyme specificity and rate. Irradiation of the polynucleotide drastically reduced the rate of hydrolysis of poly U by RNase. It was observed that RNase could not split the phosphodiester bond on the 3 -OH end of uridylic acid dimer. It was also shown that dehydration of irradiated poly U was accompanied by marked phosphodiester bond breakage and degradation of the polynucleotide. 

Phospholipase C (PTC, EC 3.1.4.3) catalyzes the hydrolysis of the phosphodiester bond in phospholipids. It releases the second messenger molecule diacylglycerin (DAG) important in the signal transduction cascade and a phosphorylated headgroup . The active site of the enzyme contains three Zn ions with two of them in close proximity. Only few crystal structures are solved until now " . ... 

The covalent backbone of DNA and RNA is subject to slow, nonenzymatic hydrolysis of the phosphodiester bonds. In the test tube, RNA is hydrolyzed rapidly under alkaline conditions, but DNA is not the 2 -hydroxyl groups in RNA (absent in DNA) are directly involved in the process. Cyclic 2, 3 -monophosphate nucleotides are the first products of the action of alkali on RNA and are rapidly hydrolyzed further to yield a mixture of 2 -and 3 -nucleoside monophosphates (Fig. 8-8). 

The known catalytic repertoire of ribozymes continues to expand. Some virusoids, small RNAs associated with plant RNA viruses, include a structure that promotes a self-cleavage reaction the hammerhead ribozyme illustrated in Figure 26-25 is in this class, catalyzing the hydrolysis of an internal phosphodiester bond. The splicing reaction that occurs in a spliceosome seems to rely on a catalytic center formed by the U2, U5, and U6 snRNAs (Fig. 26-16). And perhaps most important, an RNA component of ribosomes catalyzes the synthesis of proteins (Chapter 27). 

Hydrolysis of cAMP cAMP is rapidly hydrolyzed to 5-AMP by cAMP phosphodiesterase, one of a family of enzymes that cleave the cyclic 3 5 -phosphodiester bond. 5-AMP is not an intracellular signalling molecule. Thus, the effects of neurotransmitter- or hormone-mediated increases of cAMP are rapidly terminated if the extracellular signal is removed. [Note Phosphodiesterase is inhibited by methylxanthine derivatives, such as theophylline and caffeine.3]... 

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