Phosphate monoester, hydrolysis mechanisms

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... 

Figure 4. Possible mechanisms of phosphate monoester hydrolysis by protein phosphatase 1.

Methyl-p-nitrophenyl phosphate coordinated to the two metal centers in 37 undergoes hydrolysis by a two-step addition-elimination mechanism [73]. The free phosphate hydrolyzes by a concerted mechanism. In both phosphate monoester and diester hydrolysis, the two Co(m) centers in 32 and 37 stabilize the five-coordinate phosphate species (transition state or intermediate) by bringing the phosphate and nucleophile together. This stabilization leads to a change in mechanism from dissociative to concerted for a phosphate monoester hydrolysis [96] and from concerted to stepwise for phosphate diester hydrolysis [73]. 

Figure 11 A possible mechanism for phosphate monoester hydrolysis catalyzed by PPPs. ...

Figure 12 Possible mechanisms for phosphate monoester hydrolysis catalyzed by eukaryotic (a) and prokaryotic (b) PPMs. The representation for eukaryotic PPMs is based on the crystal structure of PP2Ca bound to phosphate (PDB code 1A6Q). In the published structure the phosphate anion is not directly coordinated to the metal ions and it has been modified here to represent a catalytic complex analogous with that implicated by the crystal structure of the homologue MspP bound to phosphate (PDB code 2JFR) see text for details. The octahedral coordination of metals Ml and M2 are completed by bridging carboxylate residues Asp60 in PP2Ca, and Asp35 in MspP (not shown for clarity). Water molecules complete the octahedral coordination at third metal site of the prokaryotic MspP.

A similar reaction mechanism was proposed by Chin et al. [32] for the hydrolysis of the biological phosphate monoester adenosine monophosphate (AMP) by the complex [(trpn) Co (OH2)]2+ [trpn = tris(ami-nopropyl)amine]. Rapid cleavage is observed only in the presence of 2 equiv metal complex. It is evident from 31P NMR spectra that on coordination of 1 equiv (trpn)Co to AMP a stable four-membered chelate complex 4 is formed. The second (trpn)Co molecule may bind to another oxygen atom of the substrate (formation of 5) and provide a Co-OH nucleophile which replaces the alkoxy group. The half-life of AMP in 5 is about 1 h at pD 5 and 25 °C. 

The highest rate acceleration in the nonenzymatic hydrolysis of a phosphate monoester was reported by Chin s group [35]. In the dinuclear cobalt(III) complex 9 the metal ions are much more rigidly preorganized than in complexes 6 and 8. At pH 7 and 25 °C coordinated phenyl phosphate (PP) hydrolyzes 1011 times faster than free PP under the same conditions. There is good evidence for a reaction mechanism which has already been suggested for 2. The higher reactivity of 9 compared to 2 may be attributed to the proximity of substrate and M-OH nucleophile. 

Converging lines of evidence have led to a general acceptance of the monomeric metaphosphate mechanism for the hydrolysis of phosphate monoester monoanions. The pH rate profile for aryl and alkyl phosphate monoester hydrolyses commonly exhibits a rate maximum near pH 4. where the concentration of the monoanion is at a maximum. The proposed mechanism is based on these principal points of evidence (a) a general observation of P-O bond cleavage (b) the entropies of activation for a series of monoester monoanions are all close to zero, which is consistent with a unimolecular rather than a bi-molecular solvolysis where entropies of activation are usually more negative by 20 eu7 (c) the molar product composition (methyl phosphate inorganic phosphate) arising from the solvolysis of the monoester monoanion in a mixed methanol-water solvent usually approximates the molar ratio of methanol ... 

In contrast, the acid-catalyzed hydrolysis of alkyl selenates is A-2158. The actual species which undergoes decomposition to alcohol and sulfur trioxide is probably the zwitterion as in the case of phosphate monoester monoanions. Evidence for sulfur trioxide as the reactive initial product of the A-1 solvolysis is obtained from the product compositions arising with mixed alcohol-water solvents. The product distribution is identical to that found for sulfur trioxide solvolysis, with the latter exhibiting a three-fold selectivity for methanol. Although the above entropies of activation and solvent deuterium isotope effects do not distinguish between the conventional A-l mechanism and one involving rate-limiting proton transfer, a simple calculation, based on the pKa of the sulfate moiety and the fact that its deprotonation is diffusion controlled. 

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. 

Recently, theoretical calculations suggested that the rate of HO- attack on the neutral phosphate monoester is very fast.229 Earlier studies underestimated this rate and the present result indicates that the hydrolysis of phosphate monoesters in aqueous solution is not inconsistent with a mechanism that involves proton transfer to the phosphate oxygen followed by nucleophilic attack on the phosphorus. The hydrolysis of... 

The pH-rate profile for unbuffered hydrolysis of glyceraldehyde-3-phosphate (6-3-P) has been attributed to hydrolysis of the monoanion of the phosphate monoester at pH < 4, spontaneous formation of glyceraldehyde from the phosphate dianion at pH 7-8, and, at higher pH, hydroxide-catalysed methylglyoxal formation. Reaction of the dianion is not subject to a solvent isotope effect and is believed to occur by the irreversible ElcB mechanism whereby an enediolate intermediate, formed on rate-determining C(2) deprotonation, subsequently expels phosphate trianion by C—0 bond breaking. The diethylacetal and 2-methyl-G-3-P do not hydrolyse under the same conditions.5... 

Elimination-addition is a mechanistic pathway open to hydrolysis reactions, for example, when they are performed in a strong base and when the substrate is charged as in Scheme 7.4. A highly unstable intermediate is generated in the rate-determining first step. This mechanism is especially prevalent in the hydrolysis of phosphate monoesters,... 

Figure 1 Active site structure of PAPs and proposed mechanisms for their catal)dic hydrolysis of a phosphate monoester, involving either a terminal (Mechanism A) or bridging (Mechanism B) hydroxide. The Fel, containing the tyrosine hgand, is in the Fe(III) oxidation state. (Reprinted with permission from Ref. 9. 2002 American Chemical Society)...

Thus, even though free metaphosphate anion cannot be considered a mechanistically significant intermediate in enzyme-catalyzed phosphate monoester ester hydrolysis, since it is unlikely that an acceptor nucleophile will not be present to participate in a preassociative mechanism, it can exist in solution under appropriate solvent conditions when an acceptor nucleophile is unavailable. 

The mechanism of an actual hydrolysis reaction catalyzed by this prototype phosphomonoesterase has never been studied stereochemically. This apparent omission is presumably explained by the very low catalytic efficiency of the enzyme toward phosphorothioate monoesters as compared to phosphate monoesters (75) certainly, chiral [ O, 0]phosphorothioate 0-ester substrates already exist, and methodology is available for the configurational analysis of the chiral [ 0, 0, 0]thiophosphate that would be produced if the chiral substrate were hydrolyzed in H2. In fact, the low catalytic reactivity of phosphorothioate O-esters and the high reactivity of phosphorothioate S-esters has been explained by the enzyme utilizing nucleophilic catalysis (an associate mechanism) to achieve hydrolysis of the phosphate ester bond 40). 

Until recently, all enzymes that catalyze the cleavage of phosphate ester bonds were assumed to be phosphohydrolases, i.e., the cleavage reaction occurs by hydrolysis of a phosphodiester bond to generate ends containing a phosphate monoester and a hydroxyl group. This assumption has now been shown to be incorrect for enzymes that introduce strand breaks on the 3 -side of aldehydic abasic sites (also referred to as apurinic, apyrimidinic, or simply AP sites) during DNA repair. Despite the title of this chapter, the mechanism of the strand cleavage reaction will be briefly described. 

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