Enzymatic regeneration of ATP: II. Equilibrium studies with acetate kinase and adenylate kinase

Abstract

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 C...

Kinetic modeling and simulation of in vitro transcription by phage T7 RNA polymerase

“…Free Mg 2+ ion concentrations were calculated by an algorithm written according to a previously proposed method (Storer and Cornish-Bowden, 1976), which is based on iteratively solving ionic species mass balance equations. The dissociation constants applied in this method for complex formation between dissolved ions and low-molecularweight system components were taken from Langer et al (1977) unless otherwise noted: K HNTP ‫ס‬ 1.26 и 10 −7 M, K H 2 NTP ‫ס‬ 8.71 и 10 −5 M, K Mg 2 NTP ‫ס‬ 5.01 и 10 −2 M, K MgNTP 2 ‫ס‬ 3.98 и 10 −3 M, K MgNTP ‫ס‬ 1.15 и 10 −5 M (O'Sullivan and Perrin, 1964), K MgHNTP ‫ס‬ 1.41 и 10 −3 M (O'Sullivan and Perrin, 1964), K MgPi ‫ס‬ 1.32 и 10 −2 M (Smith and Alberty, 1956), K MgPPi ‫ס‬ 1.70 и 10 −5 M (Käpylä et al, 1995), and K Mg 2 PPi ‫ס‬ 2.14 и 10 −3 M (Käpylä et al, 1995). The employed buffer, Tris, was shown to exhibit no significant binding capacity to metal ions (Good et al, 1966).…”

Analysis of progress curves. Rate law of pyruvate kinase type I from Escherichia coli

“…The average of the relative deviation between the measured and the calculated total volume of titrant taken over all progress curves was 0.047 for the selected set in Table 2. A set of constants including the values given by Alberty (1968) yielded a value of 0.069, and a set including the constants given by Langer et al (1977) yielded 0.076. Table 3 shows the abbreviations used for the different ligands.…”

Initial-rate studies of a thermophilic glucokinase from Bacillus stearothermophilus

“…The concentrations of MgATP2-, MgADP-, protonated complexes and other ions were calculated from the total concentrations of MgCl2, ATP, ADP and other substances as described by Storer & Cornish-Bowden (1976a), by using the thermodynamic data of Phillips et al (1966) and the data cited in a paper by Langer et al (1977). Calculation showed that the MgATP2concentration was a high and nearly constant proportion (74 + 2 %) of the total ATP concentration, and that the MgADP-concentration was also a nearly constant proportion (94±1 +%) of the total ADP concentration.…”