Phosphomonoester structure

Alkaline phosphatase (AP) is a (Znn)2-containing phosphomonoester-ase that hydrolyzes phosphomonoesters (RO—POf-) at alkaline pH (7). Ser102 under the influence of one of the zinc(II) ions at the active center 1 (Fig. 2) is directly involved in phosphate hydrolysis (8). On the basis of X-ray structure and NMR studies (9), the mechanism now accepted is that the phosphate substrate, initially recognized by cooperative... 

The X-ray crystal structure of the inorganic phosphate (an inhibitor) complex of alkaline phosphatase from E. coli (9) showed that the active center consists of a Zn2Mg(or Zn) assembly, where the two zinc(II) atoms are 3.94 A apart and bridged by the bidentate phosphate (which suggests a phosphomonoester substrate potentially interacting with two zinc(II), as depicted in Fig. 2), and the Mg (or the third Zn) is linked to one atom of the zinc pair by an aspartate residue at a distance... 

While there have been a considerable number of structural models for these multinuclear zinc enzymes (49), there have only been a few functional models until now. Czamik et al. have reported phosphate hydrolysis with bis(Coni-cyclen) complexes 39 (50) and 40 (51). The flexible binuclear cobalt(III) complex 39 (1 mM) hydrolyzed bis(4-nitro-phenyl)phosphate (BNP-) (0.05 mM) at pH 7 and 25°C with a rate 3.2 times faster than the parent Coni-cyclen (2 mM). The more rigid complex 40 was designed to accommodate inorganic phosphate in the in-temuclear pocket and to prevent formation of an intramolecular ju.-oxo dinuclear complex. The dinuclear cobalt(III) complex 40 (1 mM) indeed hydrolyzed 4-nitrophenyl phosphate (NP2-) (0.025 mM) 10 times faster than Coni-cyclen (2 mM) at pH 7 and 25°C (see Scheme 10). The final product was postulated to be 41 on the basis of 31P NMR analysis. In 40, one cobalt(III) ion probably provides a nucleophilic water molecule, while the second cobalt(III) binds the phosphoryl group in the form of a four-membered ring (see 42). The reaction of the phosphomonoester NP2- can therefore profit from the special placement of the two metal ions. As expected from the weaker interaction of BNP- with cobalt(in), 40 did not show enhanced reactivity toward BNP-. However, in the absence of more quantitative data, a detailed reaction mechanism cannot be drawn. 

All of the ribitol teichoic acids so far examined are composed of chains of ribitol residues joined through phosphodiester groups at C-l and C-5. Each chain is terminated by a phosphomonoester residue, and the ribitol residues bear glycosyl and D-alanine ester substituents. Detailed structures have been proposed for the polymers from Bacillus aubtilis and Lactobacillus arabinosus, and from two strains of Staphylococcus aureus. The structure of the teichoic acid from Bacillus subtilis was the first to be established in detail the other polymers differ mainly in the nature of the glycosyl substituents. 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

The Zn(II) ion is held by the tetra8iza assembly and further coordinated by the labile pendant alcohol, which simulates Ser 102 in the enzyme. Under these conditions, the pendant alcohol is activated toward deprotonation such that its pl drops from about 15 to 7.51 (at 298 K). Above pH 7.5, deprotonation of the pendant alcohol rather than a coordinated water molecule, was shown by NMR spectral changes associated with the protons in the pendemt arm and by the crystal structure of the deprotonated Zn(II) complex (Fig. 1). Apparently, [Zn(3)]+ hydrolyzes phosphomonoesters too slowly for ease of investigation however, the reaction with a phosphodiester, bis(4-nitrophenyl) phosphate, which is sufficient to cleuify the proposed cooperative role of Ser 102 and Zn(II) in alkahne phosphatases, is considerably faster and was... 

Fig. 1 From left to right, the structures of a phosphate monoester, diester, and triester. Depending upon pH, monoesters may be neutral, monoanionic, or dianionic diesters may be neutral or anionic. The first pKa of an alkyl phosphomonoester, and the pKa of a dialkyl diester, is 2. The second pKa of an alkyl monoester is 6.8. Oxygen atoms bonded to ester groups (OR) are called bridging oxygen atoms the other oxygen atoms are nonbridging. Thus, a triester has one nonbridging oxygen atom, an ionized diester has two, and a fully ionized monoester has three.

Structure of the substrate and the reaction conditions determine the transition state for reaction with a particular nucleophile 104, 105). The extreme cases are generally described as the dissociative and associative substitution mechanisms. The fully dissociative mechanism entails the formation of monomeric metaphosphate monoanion as a discrete intermediate and was first formulated by F. H. Westheimer, who pioneered the physical organic chemistry of the hydrolysis of phosphate esters 106, 107). This mechanism is depicted in Eq. (40) and is possible only for phosphomonoesters with good leaving groups, examples of which are shown. 

Alkaline phosphatases of bacteria catalyze hydrolyses of a variety of phosphomonoesters and also act as phosphotransferases. The one from E. coli contains four Zn2+ ions per unit of molecular weight of 89,000. Much still remains to be learned of the chemistry, and the structure is unknown. 

The requirement of the dimetallic complexes to hydrolyze phosphomonoesters was demonstrated by a dizinc(II) cryptate (48) with an alkoxide-bridge (Scheme 34). Complex (48) reacted with phosphomonoesters such as mono(4-nitrophenyl) phosphate (4-NPP) at pH 5-7 in aqueous solution. It should be noted that (48) reacts with 4-NPP exclusively, not with diester, BNPP, nor triester, tris(4-nitrophenyl) phosphate (TNPP). A second-order dependence of the rate constant (first order with respect to both [(48)] and [(4-NPP)] was determined. The rate-pH profile exhibited a bell-shaped relationship with the maximum rate at pH 5.9. On the basis of X-ray crystal-structure analysis of (48) and (50), a mechanism was proposed (Scheme 34). Initiahy, two 0 donors of 4-NPP interact with the two Zn ions, which are suitably separated (3.42 A) (49). 

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