Dianions phosphoryl group

Full details on the phosphorylation of water and alcohols by 4-nitrophenyl dihydrogen phosphate and the NfC H ) - and N(CH3) -salts of its mono- and dianion have been published 146>. Phosphoryl group transfer from the monoanion and dianion is thought to proceed via the monomeric POf ion. Addition of the sterically unhindered amine quinuclidine to an acetonitrile solution containing the phosphate monoanion and tert-butanol produces t-butyl phosphate at a faster rate than does the addition of the more hindered diisopropylethylamine. This nucleophilic catalysis of the phosphorylation reaction is also explained by the intermediacy of the POf ion. 

Robinson, 1969a). It is probable that the hydrophobic nature of the phenyl groups of p-nitrophenyl diphenyl phosphate results in deep penetration of the neutral ester in the Stern layer, thus shielding the phosphoryl group from nucleophilic attack. Unlike other reactions between nucleophiles and neutral substrates catalyzed by cationic micelles (Bunton and Robinson, 1968, 1969a) and the hydrolysis of dinitrophenyl phosphate dianions in the presence of cationic micelles (Bunton et al., 1968), the catalysis of the hydrolysis of -nitrophenyl diphenyl phosphate by CTAB arises from an increase in the activation entropy rather than from a decrease in the enthalpy of activation. The Arrhenius parameters for the micelle-catalyzed and inhibited reactions are most probably manifestations of the extensive solubilization of this substrate. However, these parameters can be composites of those for the micellar and non-micellar reactions and the eifects of temperature on the micelles themselves are not known. Interpretation of the factors which affect these parameters must therefore be carried out with caution. In addition, the inhibition of the micelle-catalyzed reactions by added electrolytes has been observed (Bunton and Robinson, 1969a Bunton et al., 1969, 1970) and, as in the cases of other anion-molecule reactions and the heterolysis of dinitrophenyl phosphate dianions, can be reasonably attributed to the exclusion of the nucleophile by the anion of the added salt. 

Fig. 28 A model of the proposed role of the conserved Glu and the y-phosphoryl group in the Ras-catalyzed hydrolysis of GTP. The alternative of water attack upon a dianionic terminal phosphoryl group has also been proposed.

Ramirez and Marecek have investigated the solvolyses of 2,4-dinitrophenyl phosphate in aprotic and protic solvents and described reaction conditions that are consistent with the involvement of free metaphosphate anion 45, 46). In particular, the dianion of the reactive phosphate monoester is capable of transferring its phosphoryl group to /err-butanol whereas the monoanion of the same ester is essentially unreactive in the same reaction. Since rert-butanol is unreac-tive as a nucleophile for steric reasons, the phosphorylation of this alcohol is considered to be a diagnostic test for the involvement of the highly reactive metaphosphate anion. 

Two different models have been proposed to explain how regulatory phosphorylation sites may exert their function. Either the dianionic phosphoserine is salt-linked to an arginine or lysine residue and in doing so may help stabilize one spedfic protein conformation, or a mobile phosphoryl group may prevent an interaction between protein domains. In the latter case, one would expect the resonance to show titration behavior with a Hill coefficient similar to that of standards, which is indeed observed for some of the proteins. When salt bridges play a role, the energy involved has to be <5 kcal mol in order to observe a titration behavior. A lower pA, and an increased Hill coeffident (n > 1) can provide evidence for the presence of proximal positive charges. 

The serine group which becomes phosphorylated does not appear to possess any marked nucleophilic reactivity, nor is there any evidence that a histidine group participates as a general acid-general base catalyst. Rate constants for the nonenzymic hydrolysis of alkyl and aryl phosphate monoanions at 25° are in the range HP7 to 10-9 sec-1 (167), while the comparable alkaline phosphatase-catalyzed values (in this case they refer to dianions) are in the range 102 to 103 sec-1. Thus one has to account for a rate enhancement factor of 109 to 1012. Moreover, the... 

Studies on the reactivation of apoglycogen phosphorylase with a variety of analogs of pyridoxal phosphate have shown that the catalytic moiety is the 5 -phosphate group - only analogs with a reversibly protonatable dianion in this position have any activity In the nonactivated form of phosphorylase b, the phosphate is monoprotonated (-OPO3H ) when the enzyme has been activated, either allosterically or by phosphorylation (phosphorylase a), it is dianionic (-OPOa ). A glutamate residue in the active site acts as the proton acceptor or donor for this transition between the inactive and active forms of the cofactor. 

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. 

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