Phosphotransferases direct transfer mechanism

A few nucleotidyltransferases and phosphotransferases act by mechanisms that include a covalent nucleotidyl-enzyme or phosphoenzyme as a compulsory intermediate. A few more ATP-dependent synthetases act by such mechanisms. However, the overwhelming majority of these enzymes catalyze phosphoryl or nucleotidyl group transfer by mechanisms that entail the direct, one-step transfer of the nucleotidyl group or phosphoryl group between two substrates bound in adjacent subsites at the active site. 

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. 

An active phosphotransferase, requiring ATP and Mg, and catalyzing the phosphorylation of -nitrophenol has been demonstrated in the lOO.OOOg supernatants of gut tissues of the Madagascar cockroach (Gromphadorhina portentosa), tobacco hornworm (M. sexta) larvae and in whole houseflies (H. domestica) (21,30) in K. portentosa it appears to constitute a major pathway for phenol metabolism (21,30). Although the mechanism of phosphorylation has not been elaborated, it is probable that ATP serves as the direct donor of the phosphate moiety transferred to the phenol (Figure 5). 

Phosphotransferases and nucleotidyltransferases were last reviewed in this series 15 years ago in Volumes VIII and IX. At that time a major mechanistic question was whether these enzymes catalyze their reactions by single-displacement or double-displacement mechanisms. The two mechanisms differed chemically with respect to whether the phosphoryl or nucleotidyl group is transferred directly between two substrates, or whether the group transfer is mediated by a nucleophilic group of the enzyme in a two-step mechanism via a covalent phos-phoenzyme or nucleotidyl-enzyme. 

---