Evolution of Oxygen Isotopologues in Phosphate and Pyrophosphate during Enzyme-Catalyzed Isotopic Exchange Reactions

Inorganic pyrophosphatase (PPase) is an enzyme that catalyzes the hydrolysis of the phosphoanhydride bond in pyrophosphate (PP_i) to release inorganic phosphate (P_i) and simultaneously exchange oxygen isotopes between P_iand water. Here, we quantified the exchange kinetics of oxygen isotopes between five P_iisotopologues (P¹⁸O₄, P¹⁸O₃¹⁶O, P¹⁸O₂¹⁶O₂, P¹⁸O¹⁶O₃, and P¹⁶O₄) and water using Raman spectroscopy and ³¹P nuclear magnetic resonance (NMR) during the PPase-catalyzed ¹⁸O-¹⁶O isotope exchange reaction in P_i-water and PP_i-water systems. At a high PP_iconcentration (300 mM), hydrolysis of PP_iby PPase was predominant, and only a small fraction of PP_i(â 1%) took part in the reversible hydrolysis-condensation reaction (PP_iâ " P_i), leading to the oxygen isotope exchange between P_iand water. We demonstrated that Raman and NMR methods can be equally applied for monitoring the kinetics of the oxygen exchange between the P_iisotopologue and water. It was found that the isotope exchange determined by the spectroscopic methods was detectable as low as 0.2% ¹⁸O abundance, but the reliability below 1% was much lower. Given that high P concentrations (≥1 mM) are required in these methods, environmental application of these methods is limited to rare high P conditions in engineered and agricultural environments.