Hydrolysis of phosphoesters

In addition, the high charge density on Mg2+ ensures that it is an excellent Lewis acid in reactions notably involving phosphoryl transfers and hydrolysis of phosphoesters. Typically, Mg2+ functions as a Lewis acid, either by activating a bound nucleophile to a more reactive anionic form (e.g. water to hydroxide anion) or by stabilizing an intermediate. 

Several zinc enzymes that catalyse the hydrolysis of phosphoesters have catalytic sites, which contain three metal ions in close proximity (3-7 A from each other). These include (Figure 12.11) alkaline phosphatase, phospholipase C and nuclease PI. In phospholipase C and nuclease PI, which hydrolyse phosphatidylcholine and single-stranded RNA (or DNA), respectively, all three metal ions are Zn2+. However, the third Zn2+ ion is not directly associated with the dizinc unit. In phospholipase C, the Zn-Zn distance in the dizinc centre is 3.3 A, whereas the third Zn is 4.7 and 6.0 A from the other two Zn2+ ions. All three Zn2+ ions are penta-coordinate. Alkaline phosphatase, which is a non-specific phos-phomonoesterase, shows structural similarity to phospholipase C and PI nuclease however,... 

Figure 2. Schematic drawing of the active site of staphylococcal nuclease. Protein side chains are shown by light bonds, while the PdTp molecule is in dark. The Ca ion is shown as the large sphere below the inhibitor molecule. Also shown are the three inner sphere water ligands of the calcium ion and the water molecule bridging Glu-43 and the 5 -phosphate of the inhibitor (this bridging water is the putative nucleophile in the hydrolysis of phosphoesters) (from ref. 1).

The hydrolysis of phosphoester bond can be accelerated by several catalytic factors, including enhancement of the nucleophilicity of the 2 -oxygen (Scheme 4.3-9), stabilization of the leaving 5 oxyanion group (Scheme 4.3-8), stabilization of the pentavalent intermediate (Scheme 4.3-8), and the presence of buffers (e.g., imidazole, morpholine, and carboxylates) and divalent or trivalent metal ions (e.g., Mg +, Zn ", Ca"", Cu"+, and Fe"+) [44]. 

A number of other enzymes which catalyze the hydrolysis of phosphoesters are of biological importance. These include cyclic purine phosphodiesterase (little is known about its active site chemistry at present, but more shall be said about its biological role shortly) and the phosphatases. Acid and alkaline phosphatase catalyze the hydrolysis of phosphomonoesters to the corresponding alcohol and inorganic phosphate. Their pH optimums are 5.0 and 8.0, respectively hence their names. Both form covalent enzyme-substrate intermediates ... 

Recently, highly branched macromolecular polyamidoamine dendrimers have been prepared with Co11 bound where the metal ions have additional exchangeable coordination sites.450 These macromolecules show a capacity for catalyzing the hydrolysis of phosphate esters, presumably via intermediate bound phosphoester species. 

The macrocyclic metal centers were also effective catalysts for hydrolysis of bis(p-nitrophenyl) phosphate (BNPP) and p-nitrophenyl phosphate (NPP). DNPP has been extensively studied as a DNA model. Among various macrocyclic metal centers built on PEI, the one (26) obtained by condensation of PEI with glyoxal in the presence of Co(II) ion was particularly effective in the hydrolysis of the phosphoesters. The half-lives for spontaneous hydrolysis of BNPP and NPP are reported as 2000 years and 4 years, respectively, at neutral pHs and 25 In... 

A phosphoanhydrlde bond or other high-energy hond (commonly denoted by ) is not Intrinsically different from other covalent bonds. High-energy bonds simply release especially large amounts of energy when broken by addition of water (hydrolyzed). For Instance, the AG" for hydrolysis of a phosphoanhydrlde bond in ATP (—7.3 kcal/mol) is more than three times the AG" for hydrolysis of the phosphoester bond (red) in glycerol 3-phosphate (—2.2 kcal/mol) ... 

Here we see the final substrate-level phosphorylation in the pathway, which is catalyzed by pyruvate kinase. Phosphoenolpyruvate serves as a donor of the phospho-ryl group that is transferred to ADP to produce ATP This is another coupled reaction in which hydrolysis of the phosphoester bond in phosphoenolpyruvate provides energy for the formation of the phosphoanhydride bond of ATP. The final product of glycolysis is p)mivate. 

A detailed investigation has been reported into the cleavage of 3 - 5 -uridyluridine to form the 2, 3 -cyclic phosphate, and its isomerization to 2 5 -uridyluridine. The hydrolysis of uridine 2 -, 3 - and 5 -phosphoromonothio-ates under acidic and neutral conditions has been investigated in mild acid only hydrolysis to uridine occurs, whilst at low pH desulfurization occurs in the cases of the 2 - and 3 -thioates. The same workers have also studied the kinetics of hydrolysis and desulfurization of the diastereomeric monothio-analogues of uridine 2, 3 -cyclic phosphate under neutral or acidic conditions desulfurization competes with phosphoester hydrolysis. The hydrolysis of the 2 -thionucleoside 3 -phosphate 267 (X=SH) has been studied the predominant reaction pathway at pH 13 is the formation of the 5-phosphate whilst at pH 7-10 mostly the 2, 3 -cyclic monothiophosphate was produced. The 2 -fluorocompound 267 (X=F), which has a C-3 -endo- conformation, underwent hydrolysis ten times faster than did the deoxycompound 267 PC=H). The kinetics of hydrolysis of thymidine 5 -boranomonophosphate (269) have been studied by NMR. It was found that 269 hydrolyses slowly to thymidine and [03P-BH3 ], with the latter hydrolysing even more slowly to phosphonate and boric acid. ... 

In this framework, the emergence of new synthetic pathways could proceed in two steps, as follows. First, a (ribo)zyme capable of cleaving a new type of chemical bond - in a thermodynamically favourable reaction — would have emerged. Then, the ability to make this particular type of chemical bond could develop as a reversal of the new catalytic pathway, provided that coupling with some exergonic reaction (e.g. a phosphate group transfer or a hydrolysis of a phosphoester bond) could be established. 

Pentacoordinate species (phosphoranes) are proposed intermediates in the hydrolysis of RN A and DNA. Before such species were well accepted, chemists examined the chemistry of phosphoesters such as i as model systems. Compound i can cyclize to give phosphorane ii, although a was never. seen at room temperature. However, upon adding acetyl chloride to a solution of /, both Hi and iv are isolated. Trapping experiments such as this one give good... 

The masters of catalysis are enzymes. Enzymes are biomolecules typically based on proteins and often associated with small organic molecules or metal ions known as cofactors. In recent years it has become clear that RNA molecules can also catalyze important reactions, and such catalytic RNA molecules are referred to as ribozymes. Our focus here, however, will be on the more well known, protein-based enzymes, which mediate the overwhelming majority of biochemical transformations. These are nature s catalysts, and they can be incredibly efficient. As just one example, the hydrolysis of a phosphoester such as that used to link nucleotides together in DNA is estimated to have a half-life of hundreds of millions of years in water at neutral pH. Yet, the enzyme staphylococcal nuclease can catalyze this hydrolysis reaction with a half-life of a few minutes. Since this is a physical organic textbook, not a biochemistry textbook, we do not look at the structures of enzymes and how they are formed. Instead, we simply focus upon the mechanisms and kinetics of enzymatic catalysis. 

Exonuclease III (Exo III) of E. coli is a monomeric multifunctional enzyme (31 kDa) that catalyzes the hydrolysis of at least four different types of phosphoester bonds in dsDNA (Fig. 3.4). The main enzymatic activity of Exo III is the 3 — 5 -exonuclease activity that carries out the successive release of 5 -P-mononucleotides from the 3 ends of dsDNA. The second activity is the DNA 3 -phosphatase activity that hydrolyzes 3 -terminal phosphomonoesters. In fact, Exo III was initially discovered as a DNA 3 -phosphatase in E. coli (1,2). Exo III has a third activity which degrades the RNA strand in a DNA RNA heteroduplex, thus the RNase H activity. The fourth activity of Exo III is an AP endonuclease which cleaves phosphodiester bonds at apurinic or apyrimidinic sites. 

Li W, Rudack T, Gerwert K, Grater F, Schlitter J. Exploring the multidimensionA free energy surface of phosphoester hydrolysis with constrained QM/MM dynanucs. 

---