Formation and Cleavage of P‐O Bonds

In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from Acinetobacter johnsonii 210A

“…PK has a half-saturation constant (K m ) for ADP of 0.1 mM, which is smaller than that of AcK (K m [MgADP] ϭ 0.4 mM) and allows ATP regeneration at low concentrations of ADP. The AcP/AcK system is widely used for large-scale ATP regeneration, since AcP can be prepared relatively easily (20). Although AcP is a weaker phosphoryl donor than PEP, the major drawback is its relative instability in solution (half-life, 21 h at 25°C, pH 7) (4).…”

“…Cofactor provision for ATP-dependent enzymes can be accomplished by their direct addition in stoichiometric amounts or by the inclusion of a cofactor regeneration system. Direct cofactor addition is not only costly but can unfavorably alter reaction equilibrium, lead to an accumulation of inhibitory cofactor by-products, and complicate recovery of end products (20). For preparative-scale organic synthesis, these problems have been overcome by the development of enzymatic systems for ATP regeneration.…”

“…Both systems involve the transfer of a phosphoryl group from a high-energy donor to ADP but have characteristics (discussed later) that favor particular applications. Both AcK and PK have broad substrate specificities, allowing the regeneration of other nucleoside or 2Ј-deoxynucleoside triphosphates (i.e., GTP, UTP, CTP, and 2Ј-dATP) (20). Disadvantages of both systems relate to the stability, expense, or requisite chemical syntheses (for large-scale application) of the high-energy phosphoryl donors and product inhibition, in the case of PK, by pyruvate.…”

“…The results indicate that PPT from A. johnsonii is specific for AMP and 2-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.Enzyme-catalyzed phosphoryl transfer reactions (i.e., those which form or cleave P-O bonds) represent a viable alternative to multistep chemical phosphorylations in preparative organic synthesis (7,10,20). Many useful phosphorylating enzymes require nucleoside triphosphates as cofactors.…”

“…Enzyme-catalyzed phosphoryl transfer reactions (i.e., those which form or cleave P-O bonds) represent a viable alternative to multistep chemical phosphorylations in preparative organic synthesis (7,10,20). Many useful phosphorylating enzymes require nucleoside triphosphates as cofactors.…”