Mechanism of Phosphodiester Hydrolysis

The difference in pH is attributed to subtle structural and electronic differences in the complexes. In [Cd2(C02EtL2)(CH3C00)2] a terminally Cd(II)-bound water (pKa = 8.7) is replacing one of the ether arms, in [Cd2(C02EtH2Ll) (CH3C00)2] however the alcohol arm is bound tightly to the Cd(II) ions and thus the terminal water has to compete with the ligand donor atoms for coordination. 

Other mechanistic scenarios are possible. One would be where the alcohol arm of the ligand is actively involved in the mechanism. In this case it could be envisaged that the alcohol arm is deprotonated at pH 10.1 and acts as the primary nucleophile to hydrolyze the substrate BDNPP. The ligand is then recovered by an external water molecule and the substrate is released. A second scenario is possible where, upon deprotonation, the alkoxide acts as a general base to activate an external water molecule which then hydrolyzes the substrate. Both scenarios would be consistent with the finding of a 50 % 0 labeled DNPP (Fig. 5.24). 

In a paper by Peralta et al. a linear correlation between the pKa values attributed to deprotonation of the Fe(III)-bound water for complexes with different substituents, methyl, H, Br and NO2 respectively, was found [54], This study shows that subtle structural changes in the ligand can influence catalytic activity and the acidity of the Cd(II) ions in nucleophilic activation. 

The next chapter will introduce five Co(II) complexes that serve as phosphoesterase and metallo-p-lactamase mimics. A spectroscopic and kinetic characterization will be presented. 

Eagleson, Concise Encyclopedia Chemistry (Walter de Gruyter, New York, 1994) 

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In view of the apparent convergent evolution of mechanism in the serine and cysteine protease family, it is interesting that two phosphodiesterases that require Ca + for catalytic activity by virtue of presumed electrophilic catalysis via direct coordination to the anionic phosphoryl oxygens of the substrate have evolved conceptually similar (general basic catalysis) but structurally distinct solutions to the problem of phosphodiester hydrolysis. 

The Ce(IV) ion accelerates this process 10 fold. The proposed mechanism of DNA hydrolysis by the bimetallic cluster [Ce 2(OH)4] is schematically depicted on fig. 10. First, the phosphate residue is coordinated to the two Ce(IV) ions in [Ce 2(OH)4] + the apparent association constant between Ce(IV) and TpT was determined to be 10 M at pH 2.0 and 50 °C. As clearly evidenced by both spectroscopic and theoretical studies (cf. sects. 4.2 and 4.3), the electrons of the scissile phosphodiester linkage are strongly withdrawn by the Ce(IV) ions. Furthermore, the orbitals of this phosphate are mixed with the orbitals of the Ce(IV), and form new hybrid-orbital(s). These two factors greatly activate the phosphodiester linkage and make it highly susceptible to the attack by various nucleophiles. 

RNA bears a hydroxyl group at the 2 -position of ribose. When it is hydrolyzed, this group functions as intramolecular nucleophile, and attacks the adjacent phosphorus atom. As the result, a pentacoordinated intermediate is formed. Then, the 5 -OH of ribonucleotide is removed from the P atom in the intermediate, and 2, 3 -cyclic monophosphate is formed as the covalent intermediate. In the following step, this cychc monophosphate is hydrolyzed, and the RNA hydrolysis is completed. Thus the reaction mechanism of RNA hydrolysis is entirely different from that of DNA hydrolysis which involves intermolecular attack by external nucleophile towards the P atom. The intramolecular reaction in RNA hydrolysis is so efficient that RNA hydrolysis is far faster (10 -10 fold) than DNA hydrolysis. Nevertheless, RNA is sufficiently stable under physiological conditions, and it is not easy to Itydrolyze it efficiently without using enzymes (in the absence of catalysts, the half-life of the phosphodiester linkage in RNA is estimated to be around 1000 years). The catalysts for RNA must be enormously active. In order to hydrolyze RNA within an hour, for example, the catalyst must accelerate the reaction by 10 -fold or more. In spite of a number of attempts, sufficiently fast non-enzymatic hydrolysis of RNA was unsuccessful for a long period of time. 

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).

Polymers 23A and 23 C are analogous to RNAs. Very interestingly, they showed catalytic activities for the hydrolysis of phosphodiesters and the cleavage of nucleic acids. To elucidate the action mechanism of the hydrolysis, the acetonide (32) of polymer 23C was synthesized (Scheme 13). 

The extremely high resistance of DNA towards hydrolysis makes it very difficult to study the mechanism of the hydrolysis reaction. Therefore, DNA is replaced in many studies by more reactive compounds with a phosphodiester bond (chart 1). Bis-(p-nitrophenyl)phosphate (BNPP) is a very popular model compound for the study of the hydrolytic cleavage by nucleases. Hydrolysis of the diphosphate ester yields two equivalents of a yellow nitrophe-nolate product, the formation of which can be monitored by spectrophotometry (kmax =... 

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. 

The mechanism of phosphate ester hydrolysis by hydroxide is shown in Figure 1 for a phosphodiester substrate. A SN2 mechanism with a trigonal-bipyramidal transition state is generally accepted for the uncatalyzed cleavage of phosphodiesters and phosphotriesters by nucleophilic attack at phosphorus. In uncatalyzed phosphate monoester hydrolysis, a SN1 mechanism with formation of a (POj) intermediate competes with the SN2 mechanism. For alkyl phosphates, nucleophilic attack at the carbon atom is also relevant. In contrast, all enzymatic cleavage reactions of mono-, di-, and triesters seem to follow an SN2... 

Because of its unusual active site and its potential as a model for mammalian PC-PLC enzymes, there has been considerable interest in the mechanism of the PLC5c catalyzed hydrolysis of phospholipids. The few mechanistic points that are commonly accepted are that a water must be activated for attack on the phosphodiester linkage, the leaving group must be protonated, and the products must leave the enzyme active site (Fig. 11). These issues will now be examined. 

The role of the nucleoside triphosphate in the hydrolysis of DNA has not yet been clarified. ATP and dATP are the most effective nucleotides and only slight activity (10% or less) is observed with the other triphosphates nucleoside diphosphates are inactive. The rate of DNA hydrolysis is proportional to the ATP concentration and the ATP is converted to ADP and inorganic phosphate in the course of the reaction. Three moles of ATP are consumed for each phosphodiester bond cleaved, indicating a complex mechanism of participation of ATP in the endonucleolytic reaction. Preliminary experiments by Takagi and his colleagues indicate that the purified enzyme catalyzes an exchange of ADP with ATP in the absence of DNA, suggesting that a phospho enzyme may be an intermediate. 

Ce(IV) ions efficiently catalyse the hydrolysis of phospho monoesters in nucleotides under physiological conditions. The proposed mechanism for the hydrolysis is illustrated in (217).189 Uranyl cations (U021) catalyse the hydrolysis of aggregated and non-aggregated p-nitrophenyl phosphodiesters such as (218)/(219) and (220), respectively.190 Bis(/>-nitrophenyl) phosphate (218) hydrolysis is accelerated ca 2.8 x 109-fold by Th(IV) cations in aqueous Brij micelles.191 The reactivity of Th(IV) towards (219) and (221 R = Et, C16H33) also exceeds that of uranyl ion190 and is comparable to that of Ce(IV) and exceeds that of other metal cations. 

The hydrolysis of adenosine 3. 5 -cyclic monophosphate (cAMP) by the cobalt complexes (215) was considered here earlier,187 as was the Ce(IV)-catalysed hydrolysis of phospho monoesters in nucleotides.189 A review (ca 100 references) on current data on the mechanism of cleavage-transesterification of RNA has appeared.258 In this review special attention was focused on the two crucial steps in the hydrolysis of RNA, i.e. cleavage-transesterification and hydrolysis of the cyclic phosphodiester (Scheme 14). The catalysis of various amines for the hydrolysis of RNA has been looked at and ethylenediamine and propane-1,3-diamine are highly active under physiological conditions because they exist as the catalytically active monocation forms.259... 

The Co(m)-complex of cyden, Co(m)Cyc, is one of the most effedive synthetic catalysts discovered so far for the hydrolysis of supercoiled DNAs [59]. The hydrolytic nature of DNA cleavage by the Co(m) complexes of polyamines including cyden has been well documented [57, 58]. The mechanism illustrated in 25 has been proposed [57] for the catalytic action of the Co(m) complexes. Given the remarkable enhancement of proteolytic activity of Cu(n)Cyc upon attachment to PCD [49], we tested the activity of Co(m)Cyc in phosphodiester hydrolysis to see if it is also enhanced greatly upon attachment to PCD derivatives [61, 62]. 

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. 

Sun W, Pertzev A, Nicholson AW. Catalytic mechanism of Escherichia coli ribonuclease 111 kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis. Nucleic Acids Res. 2005 33(3) 807-815. 

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