Equilibria of reactions catalyzed by acetate kinase and adenylate kinase were studied experimentally and theoretically. Nucleotide conversions in excess of 90% were obtained with reactants (acetyl phosphate and one nucleotide) in stoichiometric proportion for ATP regeneration from either ADP via acetate kinase or AMP via the coupled enzyme system. Observed equilibrium constants, measured as a function of [Mg*+] at pH 7.4, ranged from about 1 to 9 for the reaction catalyzed by adenylate kinase and from about 50 to 400 for the reaction catalyzed by acetate kinase. These results agreed well with prediction of a theoretical model for the multiple equilibria between all species present in solution. Theoretical calculations showed that magnesium ion complexes and totally dissociated anions are the predominant species in solution at pH 7 and [Mg2+] F 10-3 M.
SCOPEProspects for cell free enzyme cataIyzed syntheses on an industrial scale depend upon development of a practical process for regenerating adenosine 5'-triphosphate (ATP) from adenosine 5'-diphosphate (ADP) or adenosine 5'-monophosphate (AMP). A promising enzymatic route involves two reactions catalyzed by phosphotransferasesAcetate where acetyl phosphate (AcP) is the ultimate phosphate donor, and acetate (Ac) is the by-product. Both enzymes require a divalent cation (preferably Mg2+) for activity. Reaction (2) suffices for regeneration from ADP; both reactions are required for regeneration from AMP.In this paper we report equilibrium studies of reactions (1) and (2). Assays were developed to measure the concentration of each individual reactant and product in the presence of all other components and both enzymes. Observed equilibrium constants were measured for each Correspondence conrerning this paper should be addressed to C . K. experiments were conducted were selected to fall within the range where the enzymes retained substantial catalytic activity. The equilibrium composition of nucleotides was measured for ATP regeneration from AMP or ADP at pH 7.4 with the total concentrations of adenosine and magnesium held constant while acetyl phosphate concentration was varied.Under typical reaction conditions, each component may exist in a variety of forms, including totally dissociated anions, partially protonated species, divalent metal ion (Mg2+) complexes, and, under certain conditions, monovalent metal ion complexes. Reactions (1) and (2) therefore constitute a complicated network of reactions among a large variety of species, none of which is individually accessible to direct concentration meawrement.A theoretical model is presented for the multide equilibria between individual species; it permits calculation of the equilibrium comnosition given an arbitrary feed to a reactor. In developing the model it was necessary to determine which species should be incorporated 3nd to select from the literature a consistent set of stability and ionization constants which gave good agreement between prediction and data.AlChE Journal (Vol. 23, No. 1)
January, 1977Page 1
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