No abstract

To evaluate the proficiency of phosphatases as catalysts, the rate of the uncatalyzed hydrolysis of simple phosphate monoester dianions was estimated by extrapolating rates measured over a range of high temperatures. The rate of spontaneous hydrolysis of phenyl phosphate dianion indicates that a linear free energy relationship reported earlier is reliable for leaving groups whose conjugate acids have pKa values up to at least 10. Using Teflon reaction vessels, it proved possible to follow the hydrolysis of methyl phosphate and 3-(4-carboxy)-2,2-dimethylpropyl phosphate in strong alkali. Even in 1 M KOH, the reaction was found to be specific acid catalyzed. These results establish an upper limit for dianion reactivity, which had been overestimated earlier as a result of the leaching by alkali of silicic acid from quartz reaction vessels. The present findings indicate that the half-time for attack by water on alkyl phosphate dianions is 1.1 ؋ 10 12 years (k ‫؍‬ 2 ؋ 10 ؊20 s) at 25°C and that phosphatases involved in cell signaling and regulation produce the largest rate enhancements that have been identified thus far. Protein phosphatase-1 and inositol 1-phosphatase exceed all other known enzymes in their affinities for the altered substrates in the transition state.M ost enzyme reactions proceed with k cat ͞K m values in the neighborhood of 10 7 M Ϫ1 ⅐s Ϫ1 and appear to be similarly efficient according to that criterion. However, to assess the proficiency of an enzyme as a catalyst, and its corresponding affinity for the altered substrate in the transition state, it is necessary to compare k cat ͞K m with the rate constant of the corresponding reaction under the same conditions in the absence of a catalyst. In contrast to the relatively narrow range of values of k cat ͞K m observed for enzyme catalyzed reaction rates, the rates of these uncatalyzed reactions span a range of at least 19 orders of magnitude (1). Thus, differences in enzyme proficiency tend to reflect differences in the rates of the uncatalyzed reactions rather than the catalyzed reactions, and enzymes differ greatly from one another in their prowess as catalysts. Enzymes that catalyze the slowest reactions are of practical interest, in that they offer sensitive targets for inhibition by transition-state analogues. Their mechanisms of action are also particularly challenging to rationalize. Conspicuous among these slow reactions is the hydrolysis of monoesters of phosphoric acid.In biological systems, phosphate monoesters are cleaved by two groups of monoesterases that act by different mechanisms.One group of phosphomonoesterases, typified by bacterial alkaline phosphatase (2) and protein tyrosine phosphatases (3), uses an active-site nucleophile to displace the alcohol-leaving group from the substrate to form a phosphoryl-enzyme intermediate, which is subsequently hydrolyzed. Rate enhancement by enzymes that act through a double-displacement mechanism is not simple to analyze in terms of transition-state affinity or transition-state stabilization (...

The hollow cavities of coordination cages can provide an environment for enzyme-like catalytic reactions of small-molecule guests . We report here a new example (catalysis of the Kemp elimination -reaction of benzisoxazole with hydroxide to form 2-cyanophenolate) in the cavity of a water-soluble M8L12 coordination cage, with two features of particular interest. Firstly, the rate enhancement is amongst the largest so far observed: at pD 8.3, kcat/kuncat is 2 x 10 5 , due to the accumulation of a high concentration of partially desolvated hydroxide ions around the bound guest arising from ion-pairing with the 16+ cage. Secondly, the catalysis is based on two orthogonal interactions: (i) hydrophobic binding of benzisoxazole in the cavity, and (ii) polar binding of hydroxide ions to sites on the cage surface, both of which were established by competition experiments. Hundreds of turnovers occur with no loss of activity due to expulsion of the hydrophilic, anionic product. 2Since the realisation that hollow molecular container molecules can accommodate guest molecules in their central cavities, 1-3 their ability to modify the reactivity of their bound guests has been of great interest. [4][5][6][7][8] Well known examples include Cram's stabilisation of highly-reactive cyclobutadiene; 4 Fujita's 'ship-in-a-bottle' synthesis of cyclic silanol oligomers; 5 Nitschke's stabilisation of P4 in a tetrahedral cage; 6 and the demonstration of unusual regioselectivity in a Diels-Alder reaction when the two reacting molecules are co-confined in a host cavity. 7 The ultimate expression of this behaviour is efficient catalysis of a reaction occurring in the cavity of a container molecule. 8 These synthetic systems have the potential to achieve the selectivity and catalytic rate enhancements displayed by biological systems. For example, artificial container molecules provide relatively rigid and hydrophobic central cavities that may mimic binding pockets in enzymes. These containers may be purely organic hydrogen-bonded assemblies (such as Rebek's 'softball' dimer) 9 or may be metal-ligand polyhedral coordination cages [such as Fujita's Pd6/tris(pyridyl)triazine cage]. 10 Coordination cages offer particular promise in this field because of the ease with which they can be formed by a self-assembly process from very simple component parts, using the predictable coordination geometries of metal ions to provide the three-dimensional ordering of the components which generates the necessary cavity. [1][2][3]8,11,12 In order for a container molecule to act as an efficient catalyst it needs to (i) recognise and bind the guest(s); (ii) accelerate the reaction by increasing the local concentration of reactants and/or stabilising the transition state; and (iii) expel the product to allow catalytic turnover. 8 Guest binding in cage cavities has been very well studied and is becoming a mature field, 1 to the extent that a modeling tool for quantitative prediction of guest binding has recently been reported by us. 13 For a reaction of t...

Phosphodiester linkages, including those that join the nucleotides of DNA, are highly resistant to spontaneous hydrolysis. The rate of water attack at the phosphorus atom of phosphodiesters is known only as an upper limit, based on the hydrolysis of the dimethyl phosphate anion. That reaction was found to proceed at least 99% by C-O cleavage, at a rate suggesting an upper limit of 10 ؊15 s ؊1 for P-O cleavage of phosphodiester anions at 25°C. To evaluate the rate enhancement produced by P-O cleaving phosphodiesterases such as staphylococcal nuclease, we decided to establish the actual value of the rate constant for P-O cleavage of a simple phosphodiester anion. In dineopentyl phosphate, C-O cleavage is sterically precluded so that hydrolysis occurs only by P-O cleavage. Measurements at elevated temperatures indicate that the dineopentyl phosphate anion undergoes hydrolysis in water with a t 1/2 of 30,000,000 years at 25°C, furnishing an indication of the resistance of the internucleotide linkages of DNA to water attack at phosphorus. These results imply that staphylococcal nuclease (k cat ‫؍‬ 95 s ؊1 ) enhances the rate of phosphodiester hydrolysis by a factor of Ϸ10 17 . In alkaline solution, thymidylyl-3 -5 -thymidine (TpT) has been reported to decompose 10 5 -fold more rapidly than does dineopentyl phosphate. We find however that TpT and thymidine decompose at similar rates and with similar activation parameters, to a similar set of products, at pH 7 and in 1 M KOH. We infer that the decomposition of TpT is initiated by the breakdown of thymidine, not by phosphodiester hydrolysis.DNA hydrolysis ͉ DNA stability ͉ nuclease ͉ rate enhancement ͉ phosphate ester P hosphoric acid diesters are, in general, exceedingly unreactive in water (1-3), so that the phosphodiester linkages that join the nucleotides of DNA are highly resistant to spontaneous hydrolysis. By extrapolation of earlier model experiments at elevated temperatures, the uncatalyzed hydrolysis of dimethyl phosphate in neutral solution was found to proceed with an estimated rate constant of Ϸ2 ϫ 10 Ϫ13 s Ϫ1 at 25°C, corresponding to a half-time of 140,000 years. That reaction was found to proceed at least 99% by C-O cleavage, suggesting an upper limit of Ϸ1 ϫ 10 Ϫ15 s Ϫ1 at 25°C on the rate constant for spontaneous P-O cleavage of a phosphodiester anion, the reaction that is catalyzed by many phosphodiesterases (4).More recently, a rate constant of 6 ϫ 10 Ϫ7 s Ϫ1 has been reported for the decomposition of thymidylyl-3Ј-5Ј-thymidine (TpT) at 80°C in 1 M KOH (5). Extrapolation of the results obtained earlier for dimethyl phosphate hydrolysis in neutral solution (4), to 80°C, would indicate a rate Ϸ10 5 -fold slower. That discrepancy might indicate a major role for catalysis by hydroxide, but the hydrolysis of another dialkyl phosphodiester, bis-3-(4-carboxyphenyl)neopentyl phosphate (Np* 2 P), in which ␥-branching of the leaving alcohol prevents C-O cleavage (Fig. 1), also proceeds Ϸ10 5 -fold more slowly in 1 M KOH (6).In an effort to resolve that discre...

Understanding phosphoryl and sulfuryl transfer is central to many biochemical processes. However, despite decades of experimental and computational studies, a consensus concerning the precise mechanistic details of these reactions has yet to be reached. In this work we perform a detailed comparative theoretical study of the hydrolysis of p-nitrophenyl phosphate, methyl phosphate and p-nitrophenyl sulfate, all of which have served as key model systems for understanding phosphoryl and sulfuryl transfer reactions, respectively. We demonstrate the existence of energetically similar but mechanistically distinct possibilities for phosphate monoester hydrolysis. The calculated kinetic isotope effects for p-nitrophenyl phosphate provide a means to discriminate between substrate- and solvent-assisted pathways of phosphate monoester hydrolysis, and show that the solvent-assisted pathway dominates in solution. This preferred mechanism for p-nitrophenyl phosphate hydrolysis is difficult to find computationally due to the limitations of compressing multiple bonding changes onto a 2-dimensional energy surface. This problem is compounded by the need to include implicit solvation to at least microsolvate the system and stabilize the highly charged species. In contrast, methyl phosphate hydrolysis shows a preference for a substrate-assisted mechanism. For p-nitrophenyl sulfate hydrolysis there is only one viable reaction pathway, which is similar to the solvent-assisted pathway for phosphate hydrolysis, and the substrate-assisted pathway is not accessible. Overall, our results provide a unifying mechanistic framework that is consistent with the experimentally measured kinetic isotope effects and reconciles the discrepancies between theoretical and experimental models for these biochemically ubiquitous classes of reaction.