Phosphate ester, hydrolysis mechanisms

Wladkowski, B.D., L.A. Svensson, L. Sjolin, J.E. Ladner, and G.L. Gilliland. 1998. Structure (1.3 A) and charge state of a ribonuclease A-uridine vanadate complex Implications for the phosphate ester hydrolysis mechanism. J. Am. Chem. Soc. 120 5488-5498. 

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... 

Fig. 3 Mechanisms for enzymatic supramolecular polymerisation (a) Formation of supramolecular assembly via bond cleavage, (b) Formation of supramolecular assemblies via bond formation. Examples are shown of biocatalytic supramolecular polymerisation of aromatic peptide amphiphiles via (i) phosphate ester hydrolysis, (ri) alkyl ester hydrolysis, and (iii) amide condensation or reversed hydrolysis using protease...

The two-step mechanism of phosphate ester hydrolysis by the (Znn)2-containing alkaline phosphatase (AP) (7) is thus somewhat mimicked by 24. The phosphoryl intermediate 25 is generated by nucleophilic attack of the alkoxide moiety in 24b at BNP" and is hydrolyzed by the intramolecular Zn11—OH" species in 25b. Thus, the attack at the BNP... 

There have been a few reports of first generation coordination complex structural models for the phosphatase enzyme active sites (81,82), whereas there are some examples of ester hydrolysis reactions involving dinuclear metal complexes (83-85). Kim and Wycoff (74) as well as Beese and Steitz (80) have both published somewhat detailed discussions of two-metal ion mechanisms, in connection with enzymes involved in phosphate ester hydrolysis. Compared to fairly simple chemical model systems, the protein active site mechanistic situation is rather more complex, because side-chain residues near the active site are undoubtedly involved in the catalysis, i.e, via acid-base or hydrogenbonding interactions that either facilitate substrate binding, hydroxide nucleophilic attack, or stabilization of transition state(s). Nevertheless, a simple and very likely role of the Lewis-acidic metal ion center is to... 

The overall efficiency of these nonmolecular bimetallic sysems to promote phosphate ester hydrolysis was illustrated with ApA as RNA model (0.1 mM) hydrolysis occurred with a mixture of LaCls and FeCls (10 mM each) to more than 70% at 50°C and neutral pH in 5 min (351). The products of the reaction included adenosine and 2 -and 3 -monophosphate adenonsine, indicating that the mechanism involved a first trans-esterification step by the vicinal 2 -OH of ribose. With the same mixture of metal ions, the DNA model TpT (0.1 mM) (Fig. 18), left, B = thymine) was converted to thymidine and 3 - and 5 -monophosphate th5miidine with 36% of conversion in 24 hr at 70°C at neutral pH. The important point is the notion of cooperativity between the two metals. 

The dependence of the activity of calcineurin on the redox state of the metal center highlights its importance for catalysis and provides clues to its function in that process. Site-directed mutagenesis studies of PPl, calcineurin, and bacteriophage X protein phosphatase have also provided insights regarding the roles of non-ligand active site residues. Furthermore, the contributions afforded by studies of synthetic model compounds which mimic features of metallophosphatase active sites provide important clues to possible catalytic mechanisms. Indeed many of these models exhibit impressive rate enhancements for phosphate and phosphonate ester hydrolysis [54-62]. In this section we discuss current models regarding the mechanism of phosphate ester hydrolysis by calcineurin and other metallophosphatases in consideration of these studies. 

Figure 17-11 Two-step mechanism for phosphate ester hydrolysis. In the first step, substrate binds to the Fe ion, thereby displacing the bond from the -hydroxo ligand to this metal ion. The resulting Fe -coordinated hydroxide serves as the nucleophile in the hydrolysis reaction. The resulting species is a phosphate bridge, the same species observed in x-rays studies of product-inhibited enzyme.

Florian, J. and Warshel, A. (1998) Phosphate ester hydrolysis in aqueous solution associative versus dissociative mechanisms. Journal of Physical Chemistry B 102, 719-734. 

The use of a mixed-valent, dinuclear iron site, similar to those in hemerythrin and ribonucleotide reductase,to catalyze a nonredox reaction such as phosphate ester hydrolysis is novel and unexpected for a variant of the familiar oxo(hydroxo)-bridged diiron center. In contrast to the general agreement that exists regarding the spectroscopic and physical properties of the PAPs, their kinetics properties and especially their mechanism of action remain controversial. Much of the disagreement stems from the different pH dependences of the catalytic activity of BSPAP and Uf, which is due to the fact that the former is isolated in a proteolytically activated form while the latter is not. Proteolysis results in a substantial increase in optimal pH in addition to an increase in catalytic activity at the optimal pH. "" Current data suggest that many of the spectroscopic studies described in the literature were performed on a catalytically inactive form of the enzyme. As a result, the roles of the trivalent and divalent metal ions in catalysis and in particular the identity of the nucleophilic hydroxide that directly attacks the phosphate ester remain unresolved. 

The three most likely mechanistic possibilities for catalysis of phosphate ester hydrolysis by PAPs are shown in Figure 14. Each mechanism utilizes a different hydroxide as the nucleophile a... 

Fe Fe or Fe Zn forms, despite the 100-fold lower rate of ligand exchange for A1 Fe3+ i 4 Regardless of which mechanism is correct, the ability to replace Fe " " by Zn " " and vice versa, with full retention of activity, suggests that it is likely to be applicable to both the dinuclear iron site in the mammalian PAPs and the FeZn site in the plant PAPs. In all cases, the key feature appears to be the use of a trivalent-divalent dinuclear site for phosphate ester hydrolysis. 

Various mechanisms of phosphate ester hydrolysis have been discussed in the literature (3.98) to (3.100). 

The availibility of the three isomers of P-cyclodextrin bisimidazole (6, 7, and 8) gives us the opportunity to examine other bifunctional catalyses. Geometric preferences among the three isomers should also give us detailed information about the mechanisms, just as it did for the phosphate ester hydrolysis discussed above. The first reactions we have examined (Scheme 7) concern enolization of a ketone and processes that ensue. 

In 1979, Kluger s group examined in more detail his mechanism of urea participation in phosphate ester hydrolysis (334). In addition to contributing further evidence for the potential involvement of 0-phosphobiotin... 

Barab s, 0.> Pongracz, V., Kov ri, J.> Wilmanns, M.> 8c Vertessy, B. G. (2004). Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase. The Journal of Biological Chemistry, 279, 42907. 

Berente, I., Beke, T., 8c Ndray-Szab6, G. (2007). Quantum mechanical studies on the existence of a trigonal bipyramidal phosphorane intermediate in enzymatic phosphate ester hydrolysis. Theoretical Chemistry Accounts, 118,129. 

Klahn M, Rosta E, Warshel A. On the mechanism of hydrolysis of phosphate monoesters dianions in solutions and proteins. J Am Chem Soc. 2006 128 15310-15323. Florian J, Warshel A. A fundamentA assumption about OH- attack in phosphate ester hydrolysis is not fuUy justified. J Am Chem Soc. 1997 119 5473-5474. 

Perhaps the most extensively studied catalytic reaction in acpreous solutions is the metal-ion catalysed hydrolysis of carboxylate esters, phosphate esters , phosphate diesters, amides and nittiles". Inspired by hydrolytic metalloenzymes, a multitude of different metal-ion complexes have been prepared and analysed with respect to their hydrolytic activity. Unfortunately, the exact mechanism by which these complexes operate is not completely clarified. The most important role of the catalyst is coordination of a hydroxide ion that is acting as a nucleophile. The extent of activation of tire substrate througji coordination to the Lewis-acidic metal centre is still unclear and probably varies from one substrate to another. For monodentate substrates this interaction is not very efficient. Only a few quantitative studies have been published. Chan et al. reported an equilibrium constant for coordination of the amide carbonyl group of... 

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. 

B. Solvolysis of Phosphoric Acid Derivatives.—Interest continues in neighbouring-group participation in the solvolysis of phosphate esters. As a potential model compound for investigating the mechanism of ribo-nuclease action, the phenyl hydrogen phosphate ester of c/j-3,4-tetrahydro-furandiol (24) has been the subject of a detailed study. Above (and probably also below) pH 4 hydrolysis gives solely the cyclic phosphate (25)... 

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