Phosphorylation monoesters from

The involvement of monomeric metaphosphate in the phosphoryl transfer from phosphate monoesters, and of pentaco-ordinate intermediates from phosphotriesters represent two extremes in the mechanistics of the phosphoryl transfer process. Between the extremes are the (S 2)p processes involving transition states having various bond orders, but no true... 

In any reaction where the cleavage of a carbon-hydrogen bond is important, the introduction of a metal ion into the molecule in the proper position will facilitate reaction. For example, in the elimination of the elements of a phosphoric acid monoester from the molecule below, the electrostatic attraction of the cupric ion facilitates removal of the proton on the o -carbon atom with subsequent elimination of the phosphoryl residue (8). 

Certain dipositive metal ions not only accelerate the hydrolysis of ATP, as mentioned above, but also assist in the transfer of a phosphoryl group from one molecule to another. Such a nonenzymatic transphosphorylation takes place between ATP and an orthophosphate ion or its monoester in the presence of calcium(II), cadmium(II), and manganese(II). This type of nonenzymatic process can serve as a model for the biologically important enzymatic transphosphorylations that involve ATP, although... 

A later study in a hard-water meso-trophic lake also investigated the separate fate of i C- and P-labelled glucose 6-phosphate into bacterial-sized particles and phytoplankton (Hernandez et al., 1996). Aquatic bacteria and phytoplankton assimilated the phosphoryl moiety 100 times faster than they assimilated the glucosyl moiety. Bacterial uptake conformed to Michaelis-Menten kinetics with an apparent K, of 86 nM and a of 1.4 nM/min. Phytoplankton took up phosphoryl with an apparent K of 380 nM and a V of 7.6 nM/min. These investigators determined the concentration of naturally occurring phosphate monoesters to be between 25 and 40 nM. Comparison of the rate of phosphoryl uptake from glucose 6-phosphate with the phosphate uptake rate indicated that phytoplankton could satisfy a significant portion (42-99%) of their phosphorus demand by hydrolysis of phosphate monoesters. A similar comparison indicated that bacterial assimilation of phosphorus from... 

Given the fact that alkaline phosphatase will catalyze the stereospecific (retention) transfer of a chiral [ 0, 0, 0]phosphoryl group from any monoester to the primary hydroxyl group of 1,2-propanediol (Jones et al., 1978), this method of configurational analysis for chiral monosters can be considered general. 

Fig. 2. Synthesis of uma2enil (18). The isonitrosoacetanihde is synthesized from 4-f1iioroani1ine. Cyclization using sulfuric acid is followed by oxidization using peracetic acid to the isatoic anhydride. Reaction of sarcosine in DMF and acetic acid leads to the benzodiazepine-2,5-dione. Deprotonation, phosphorylation, and subsequent reaction with diethyl malonate leads to the diester. After selective hydrolysis and decarboxylation the resulting monoester is nitrosated and catalyticaHy hydrogenated to the aminoester. Introduction of the final carbon atom is accompHshed by reaction of triethyl orthoformate to...

Phosphorylation of cholesterol followed by the normal hydrolytic work-up gives the phosphate monoester, not the symmetrical pyrophosphate diester as previously claimed. Cholesteryl phosphorodichloridate and some related steroidal phosphorodichloridates have been prepared from the action of pyrophosphoryl chloride on the appropriate alcohol ... 

With [32P]phosphoric diimidazolide, prepared from sulfinyldiimidazole or CDI and H332P04, the 5 -terminal monoester phosphate groups in various RNAs could be selectively phosphorylated (radioactive labeling method) 1571,1581... 

If monomeric 151 is generated from the phosphonic monoester 171 (OH in place of Oe) in the presence of 2,2,6,6-tetramethylpyridine as base, then it also adds to carbonyl compounds U9,120). Thus acetophenone is smoothly phosphorylated to the corresponding enol phosphate in 90% yield. 

Although the metaphosphate mechanism for hydrolysis is well documented, such a pathway remains to be demonstrated in a biological system. Our present knowledge of many enzymic reactions allows, at best, the formulation of a preliminary mechanism, i.e. the chemical identity of substrates and enzymic intermediates and the minimal kinetic scheme. For example, much recent attention has been focused on the remarkable stability of the covalent phos-phoryl-enzyme (an O-phosphoryl serine) derived from E. coii alkaline phosphatase28 and inorganic phosphate, and on a systematic kinetic study of the enzyme s substrate specificity (O-, N- and S-monoesters) -9. Dephosphorylation of the enzyme, however, does not appear to be via a metaphosphate mechanism30. 

The whole question of the specificity was reopened with the discovery that E. coli phosphatase, contrary to an earlier statement (114), hydrolyzed a variety of polyphosphates including metaphosphate of average chain length 8 (97). It was subsequently reported that partially purified phosphatases from several mammalian tissues had appreciable PPi-ase activity at pH 8.5 (115). This was confirmed (116) and extended to include ATPase and fluorophosphatase activities (117). Proof that the same enzyme is responsible for the monoesterase and PPi-ase activities was afforded by heat inactivation studies, cross inhibition experiments, and inhibition of PPi-ase activity by L-phenylalanine, a specific inhibitor of intestinal phosphatase. It was also found that calf intestinal phosphatase couid be phosphorylated by 32P-PP and the number of sites so labeled agreed with the number of active sites determined with a monoester substrate using a stopped-flow technique (118). It would seem that the main reason for the confusion with regard to the PPi-ase activity results from the inclusion of Mg2+ in the assay. This stimulates the monoesterase activity but almost completely inhibits PPi-ase activity (117). 

A detailed study of the specific rates of solvolysis of N,N,N, N -tetra-methyldiamidophosphorochloridate (80) (TMDAPC) with analysis in terms of the extended Grunwald-Winstein equation has been reported (Scheme 19). The stereochemistry of nucleophilic attack at tetracoordinate phosphorus was also discussed." The initial reaction of bis (2,4-dinitrophenyl) phosphate (BDNPP) (81) with hydroxylamine involves release of 1 mol 2,4-dinitrophen-oxide ion and formation of a phosphorylated hydroxylamine (82), which reacts readily with further NH2OH, giving the monoester (83). The intermediate (82) also breaks down by two other independent reactions one involves intramolecular displacement of aryloxide ion (83) and the other involves migration of the 2,4-dinitrophenyl group from O to N and formation of phosphorylated 2,4-dinitrophenylhydroxylamine (84) (Scheme 20)." ... 

Phosphoryl transfer reactions have essential roles throughout biochemistry. The enzymes that catalyze these reactions result in tremendous rate enhancements for their normally unreactive substrates. This fact has led to great interest in the enzymatic mechanisms, and debate as to whether the mechanisms for enzyme-catalyzed hydrolysis of phosphate esters differ from those of uncatalyzed reactions. This review summarizes the uncatalyzed reactions of monoesters, diesters and triesters. A selection of enzymatic phosphoryl transfer reactions that have been the most studied and are the best understood are discussed, with examples of phosphatases, diesterases, and triesterases. 

Hydroxysteroids and 17/8-hydroxysteroids can be directly phosphorylated to monoesters with phosphoryl chloride in good yield. The intermediate dichloride from androstenolone has been characterized. 

A/B Phosphatases Phosphoryl group transfer from a phosphoric monoester to water as an acceptor molecule. (Phosphoric monoesters are cleaved hydrolytically). 3.1.3. 3.6.1. Phosphoric ester hydrolases Hydrolases acting on acid anhydrides in phosphorous-containing anhydrides... 

The stereochemical consequences of the methanolyses of the monoanion of phenyl [ 0, 0, 0]phosphate, the dianion of 4-nitrophenyl [" 0, 0, 0] phosphate, and [ 0, 0, 0]phosphocreatine have been determined by Knowles and co-workers (35). The monoanion of phenyl phosphate behaves as a typical phosphate monoester in that its rate of hydrolysis is maximal at pH 4, where an intramolecular proton transfer is possible. The dianion of 4-nitrophenyl phosphate is highly reactive since protonation of the leaving group is not necessary. Finally, A-phosphoguanidines have been reported to be the most reactive phosphoryl compound (the chiral phosphocreatine can be enzymically synthesized from [ y- 0, 0, 0]ATP). Thus, the solvolyses of all three of these compounds are believed to involve the participation of metaphosphate anion. The meth-anolysis of each of these compounds proceeds with quantitative inversion of configuration. 

Stereochemistry is another powerful tool for determining the net reaction pathway of phosphatases and sulfatases. These enzymes catalyze the net transfer of a phosphoryl or sulfuryl group to water from a monoester, producing inorganic phosphate or sulfate. Inversion results when the reaction occurs in a single step (Scheme 2, pathway a). Phosphatases that transfer the phosphoryl group directly to water with inversion typically possess a binuclear metal center and the nucleophile is a metal-coordinated hydroxide. Examples of phosphatases that follow this mechanism are the purple acid phosphatases (PAPs) and the serine/threonine phosphatases (described in Sections 8.09.4.3 and 8.09.4.4.1). Net retention of stereochemistry occurs when a phosphorylated or sulfiirylated enzyme intermediate is on the catalytic pathway, which is hydrolyzed by the nucleophilic addition of water in a subsequent step (Scheme 2, pathway b). 

This would be analogous to the change that results from alkyl substitution that is, transition states become more associative in the continuum from monoesters to triesters. Although relatively few phosphatases have been subjected to serious scrutiny of their transition states, in the cases that have been reported, this prediction has not been borne out. The reactions catalyzed by AP proceeds through loose transition states that are not significantly altered from those in solution, both in its phosphatase and in its promiscuous sulfatase activities. " Results with A-phosphatase and with calcineurin, which both catalyze phosphoryl transfer to a metal-coordinated hydroxide nucleophile, also provide no evidence of a significantly different transition state. Protein tyrosine phosphatases (PTPs), which do not contain metal ion cofactors but have a conserved arginine residue and proceed via a phosphocysteine intermediate, similarly catalyze phosphoryl transfer via a transition state similar to the one in solution. ... 

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