Phosphomonoester

Attempts to separate the isomeric deoxy ribose phosphates by ion exchange techniques met with no success this may be because of the fact that eluents having a pH lower than 5 were not used, as migration of phosphomonoesters is known to be acid catalysed, and, because of the free sugar involved, alkaline eluents could not be considered. 

AP isoenzymes can cleave associated phosphomonoester groups from a wide variety of substrates. The exact biological function of these enzymes is not well understood. They can behave in vivo in their classic phosphohydrolase role at alkaline pH, but at neutral pH AP isoenzymes can act as phosphotransferases. In this sense, suitable phosphate acceptor molecules can be utilized in solution to increase the reaction rates of AP on selected substrates. Typical phosphate acceptor additives include diethanolamine, Tris, and 2-amino-2-methyl-lpropanol. The presence of these additives in substrate buffers can dramatically increase the sensitivity of AP ELISA determinations, even when the substrate reaction is done in alkaline conditions. 

Carbodiimide modification of the phosphomonoester end groups on DNA molecules was first used in Khorana s lab to determine nucleotide sequences (Ralph et al., 1962). That early... 

Multinuclear MRS studies have demonstrated alterations in brains of schizophrenic patients. Phosphorus MRS studies, looking at both first-onset-unmedicated and chronic-medicated schizophrenia, consistently report alterations in the phospholipid profile in the frontal and temporal cortex [26-30]. Typically, phosphomonoester (PME) levels are lower than in controls, and phosphodiesters... 

The results obtained with this approach are quite impressive. Large rate accelerations (kcat/kuncat = 3 X 104) were observed with the best of the catalysts. A comparison between the best catalyst and a catalytic antibody system designed for phosphomonoester hydrolysis is reported. The combinatorial derived system gives an observed rate constant that is five times larger than that reported for the antibody system. In a control experiment, it was determined that polymers with just one type of carboxylic acid attached did not have catalytic activity. It... 

Phosphotransferases with an alcohol other than 5 -phosphomonoesters... 

A. S. Kearney, V. J. Stella, The in vitro Enzymic Labiloties of Chemically Distinct Phosphomonoester Prodrugs , Pharm. Res. 1992, 9, 497-503. 

E. J. Mclntee, R. P. Remmel, R. F. Schinazi, T. W. Abraham, C. R. Wagner, Probing the Mechanism of Action and Decomposition of Amino Acid Phosphomonoester Amidates of Antiviral Nucleoside Prodrugs , J. Med. Chem. 1997, 40, 3323-3331. 

Enzymes catalyzing cleavage of DNA, including endo-deoxyribonucleases that generate 5 -phosphomono-esters [EC 3.1.21.x], endodeoxyribonucleases that produce products other than 5 -phosphomonoesters [EC 3.1.22.x], site-specific endodeoxyribonucleases acting on altered bases [EC 3.1.25.x], and exodeoxyribonucleases producing 5 -phosphomonoesters [EC 3.1.11.x]. A few examples are ... 

Inositol-1,4,5-trisphosphate 5-phosphatase [EC 3.1.3.56], also known as inositol trisphosphate phosphomonoester-ase and inositol polyphosphate 5-phosphatase, catalyzes the hydrolysis of D-myo-inositol 1,4,5-trisphosphate to produce D-myo-inositol 1,4-bisphosphate and orthophosphate. The type I enzyme (but not the type II enzyme) will also hydrolyze inositol 1,3,4,5-tetrakisphosphate at the 5-position. However, neither of the two... 

This enzyme [EC 3.1.26.3], also known as RNase O and RNase D, catalyzes the endonucleolytic cleavage of RNA to 5 -phosphomonoesters. The enzyme cleaves multimeric tRNA precursors at the spacer region and is also involved in the processing of precursor rRNA, hnRNA, and early T7-mRNA. This enzyme can also act on double-stranded DNA. 

K. Hida, I. L. Kwee and T. Nakada, Ti values of phosphomonoester and phosphocrea-tine of brain show no significant change during development. Magn. Reson. Med., 1992, 27,179-182. 

R. F. Deicken, M. W. Weiner and G. Fein, Decreased temporal lobe phosphomonoesters in bipolar disorder. /. Affect. Disord., 1995, 33,195-199. 

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... 

Fig. 2. Phosphomonoester hydrolysis at active center of alkaline phosphatase.

M aqueous NaOH (done quickly before the subsequent hydrolysis could occur to any extent) showed the monodeprotonation with pKa value of 9.1, which was assigned to the 25a = 25b equilibrium. The pK value was higher than that of 7.3 for 24a under the same conditions, which is ascribable to the proximate phosphate anion interaction with zinc(II) (like 25c). The pendent phosphodiester in 25b underwent spontaneous hydrolysis in alkaline buffer to yield a phosphomonoester-pendent zinc(II) complex 26. Plots of the first-order rate constants vs pH (=7.5 -10.5) gave a sigmoidal curve with an inflection point at pH... 

The iron(II)-iron(III) form of purple acid phosphatase (from porcine uteri) was kinetically studied by Aquino et al. (28). From the hydrolysis of a-naphthyl phosphate (with the maximum rate at pH 4.9) and phosphate binding studies, a mechanism was proposed as shown in Scheme 6. At lower pH (ca. 3), iron(III)-bound water is displaced for bridging phosphate dianion, but little or no hydrolysis occurs. At higher pH, the iron(III)-bound OH substitutes into the phosphorus coordination sphere with displacement of naphthoxide anion (i.e., phosphate hydrolysis). The competing affinity of a phosphomonoester anion and hydroxide to iron(III) in purple acid phosphatase reminds us of a similar competing anion affinity to zinc(II) ion in carbonic anhydrase (12a, 12b). 

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. 

Fig. 10. A possible mechanism for phosphomonoester hydrolysis at the catalytic site of serine/threonine phosphatase-1 active site.

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