Enzymic Phosphate Ester Hydrolysis

The 3, 5 -cyclic monophosphate of adenosine (cAMP) (2.148) is an important secondary messenger for intercellular communication in biochemistry. When the cell is stimulated by the first messenger, compound 2.148 is formed from adenosine triphosphate (ATP) (Scheme 2.25). This reaction is catalysed by an adenosine cyclase enzyme. The cAMP then goes on to activate other intracellular enzymes, so producing a cell response. The response is terminated by the hydrolysis of cAMP by phosphodiesterase (a phosphate-ester-hydrolysis enzyme). The action of adenylate cyclase has been mimicked successfully with a p-cyclodextrin complex of Pr(iii) and other lanthanide(iii) metals, under physiological conditions. The... 

Ivie 1980) and quantification of its urinary metabolites in various animal species (Bucci et al. 1992 Hart 1976 Ivie 1980 Snodgrass and Metker 1992 Weiss et al. 1994). Hydrolysis of one of the two phosphate ester bonds liberates isopropanol and converts diisopropyl methylphosphonate to IMPA. The locations of the enzymes capable of catalyzing diisopropyl methylphosphonate phosphate ester hydrolysis have not been identified. 

The higher coordinating ability and Lewis acidity of Zn(H) ion in addition to the low pK of the metal-bound water molecule and the appearance of this metal ion in native phosphatases inspired a number of research groups to develop Zn(II)-containing dinuclear artificial phosphatases. In contrast, very few model compounds have been published to mimic the activity of Fe(III) ion in dinuclear centers of phosphatase enzymes. Cu(II) or lanthanide ions are not relevant to natural systems but their chemical properties in certain cases allow extraordinarily high acceleration of phosphate-ester hydrolysis [as much as 108 for copper(II) or 1013 for lanthanide(III) ions]. 

As an explanation of the phosphoryltransferase activity of mammalian phosphatases, Morton (117) had earlier advanced the idea that phosphate ester hydrolysis catalyzed by the nonspecific phosphatases occurs in two catalytic steps similar to that shown by Wilson et al. (118) for cholinesterase. The first step is the formation of a phosphoryl enzyme with the splitting out of the alcohol group. The second step is the hydrolysis of the phosphoryl enzyme. 

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 active site similarities listed above belie a remarkable functional diversity, which includes phosphate ester hydrolysis, dioxygen and NO reduction, reversible O2 binding, and O2 activation, the last of which includes enzymes involved in ribonucleotide reduction, hydrocarbon monooxygenation, and fatty acyl desaturation. At the overall protein level, the purple acid phosphatases (PAPs) seem to be completely unrelated, both structurally and functionally, to any of the others in this class. Similarly, the flavo-diiron enzymes form a structurally and probably functionally distinct family of proteins, catalyzing both dioxygen and NO reduction. These last two examples illustrate that attempts to shoehorn all of these enzymes into a single class can sometimes provide a simplistic and misleading view of their chemistry and biochemistry. 

Spectral changes found at pH <5.5 indicate that pXai is due to a metal-bound moiety that is deprotonated in the active enzyme. A plausible candidate for such a group is a metal-bound water, which upon deprotonation could act as the nucleophile in phosphate ester hydrolysis. 

Nearly 20 years have elapsed since the subject of phosphate ester hydrolysis has been discussed in a chapter in The Enzymes (/). During that time, meth-... 

The sEH enzyme catalyzes the hydrolysis of various epoxides to their corresponding diols. Homologues of sEH have been identified in almost all organisms, including bacteria, fungi, plants, insects, and mammals. The human sEH is an interesting enzyme that possesses two different active sites, one for epoxide hydrolysis and one for phosphate ester hydrolysis." " In humans, sEH is mainly found in the liver, where it converts various xenobiotic epoxides into their corresponding vicinal diols. sEH exhibits a broad substrate specificity but seems to prefer rntw-substituted epoxides. ... 

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.

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. 

Phosphate Ester Hydrolysis Catalyzed by a Cydodextrin-based Enzyme Mimic... 

Merkx M, Averill BA. 1999. Ifrobing the role of the trivalent metal in phosphate ester hydrolysis preparation and characterization of purple acid phosphatases containing Al Zn and rn Zn active sites, including the first example of an active aluminum enzyme. JAm Chem Soc 121 6683-6689. 

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