Phosphotransferase acceptors

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. 

Phosphotransferases with paired acceptors 3.1.30 Endonucleases active with either ribo- or... 

This phosphotransferase [EC 2.7.2.1] catalyzes the thermodynamically favored phosphorylation of ADP to form ATP Aeq = [ATP][acetate]/ [acetyl phosphate] [ADP] = 3000). GDP is also an effective phosphoryl group acceptor. This enzyme is easily cold-denatured, and one must use glycerol to maintain full catalytic activity. Initial kinetic evidence, as well as borohydride reduction experiments, suggested the formation of an enzyme-bound acyl-phosphate intermediate, but later kinetic and stereochemicaT data indicate that the kinetic mechanism is sequential and that there is direct in-line phosphoryl transfer. Incidental generation of a metaphosphate anion during catalysis may explain the formation of an enzyme-bound acyl-phosphate. Acetate kinase is ideally suited for the regeneration of ATP or GTP from ADP or GDP, respectively. 

Phosphotransferases with an alcohol group as acceptor (nucleophile). 

Considerable ingenuity was required in both the synthesis of these chiral compounds695 697 and the stereochemical analysis of the products formed from them by enzymes.698 700 In one experiment the phospho group was transferred from chiral phenyl phosphate to a diol acceptor using E. coli alkaline phosphatase as a catalyst (Eq. 12-36). In this reaction transfer of the phospho group occurred without inversion. The chirality of the product was determined as follows. It was cyclized by a nonenzymatic in-line displacement to give equimolar ratios of three isomeric cyclic diesters. These were methylated with diazomethane to a mixture of three pairs of diastereoisomers triesters. These dia-stereoisomers were separated and the chirality was determined by a sophisticated mass spectrometric analysis.692 A simpler analysis employs 31P NMR spectroscopy and is illustrated in Fig. 12-22. Since alkaline phosphatase is relatively nonspecific, most phosphate esters produced by the action of phosphotransferases can have their phospho groups transferred without inversion to 1,2-propanediol and the chirality can be determined by this method. 

Treatment of the enzyme with A-bromosuccinimide oxidized 2 of the 8 tryptophan residues and 8 of the 20 tyrosine residues, but none of the histidine residues. This treatment causes the phosphotransferase activity with tris as an acceptor to double and the hydrolase activity to increase slightly. In the case of cobalt alkaline phosphatase, the above treatment caused a threefold increase in hydrolase activity and the generation of an even greater phosphotransferase activity (91). 

Two major difficulties must be considered in any assay for acid phosphatase. The enzyme is subject to surface inactivation (23, 24). Accordingly, reproducible initial hydrolytic rates are not always obtained, and the kinetic behavior should be checked in any new assay developed. Discrepancies between the amount of inorganic phosphate produced and phenol liberated from phenolic phosphates may be substantial if extensive phosphotransferase activity occurs because of phosphoryl acceptor action on the part of hydroxylic buffers or other constituents of the incubation mixture (25, 26). Fluorogenic assays have been developed with very high sensitivity (27). Reference will be made to particular assays in the discussion of the specific enzymes. 

Fig. 6. Effects of varied substrate concentrations on velocity v of phosphotransferase reactions, described in terms of conventional (136) double-reciprocal plots. In (A) v has been assessed as a function of concentration of phosphoryl donor D with phosphoryl acceptor constant at concentrations Ai, A2, Aj, or A,. In (B) v has been determined as a function of varied concentrations of phosphoryl acceptor A with phosphoryl donor held constant at concentrations Di, D2, Da, or D.. (Generalized from references Jfi, 41, and 43-)...

Generally, phosphotransferase is assayed using a-methylmannoside as acceptor. After incubation, the reaction is terminated, and the phosphodiester product of the transfer reaction, (i.e., [l4C or 3H]GlcNac-a-l-phospho-6-mannose-a-l-methyl) is chromatographically separated from the reaction components, and the radioactivity is determined. The general reaction scheme is... 

Enzymes have been classified by an international Enzyme Commission (EC) and assigned EC numbers. Thus the enzyme creatine kinase (the muscle enzyme that catalyses the energy storage reaction ATP + creatine —> ADP + phosphocreatine) has the EC number 2.7.3.2, these numbers successively referring to a transferase function (2), a phosphotransferase function (7), phosphotransfer with a nitrogen (N) acceptor (3) and creatine kinase per se (2). 

Protein phosphorylation is a specific enzymatic reaction in which one protein serves as a substrate for a protein kinase. Protein kinases are phosphotransferases. They catalyze the transfer of a phosphate group from ATP to an acceptor amino acid in the substrate protein (Fig. 2.5). A detailed discussion of protein kinases can be found in Chapter 7. 

A Phosphomutases Phosphoryl group transfer, for which the acceptor is another functional group on the donor molecule. 2.7.5. 5.4.2. Phosphomutases Intramolecular phosphotransferases... 

A/B Phosphokinases Phosphoryl group transfer Nucleoside triphosphate is 2.7.1. Phosphotransferases with an alcohol group as acceptor... 

Phosphotransfera- ses the acceptors. Compounds different than nucleoside triphosphates are the donor and some other molecules than H20 are the acceptors. 2.7.4. Phosphotransferases with a phosphate group as acceptor... 

Hexokinase has activity with D-glucose, D-mannose, and D-glucosamine as acceptors. There are phosphotransferases specific for all of the monosaccharides. 

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