Kinetics of enzyme-catalysed oxygen isotope exchange between phosphate and water revealed by Raman spectroscopy

Pyrophosphatases (EC 3.6.1.1)are ubiquitous enzymes that catalyse the hydrolysis of pyrophosphate, a byproduct of many biochemical reactions. The hydrolysis leads to an oxygen isotope exchange between the newly formed phosphate molecules and water. Here, we applied Raman spectroscopy to monitor the oxygen isotope exchange reaction in presence of pyrophosphatase from baker's yeast. For this purpose, enzymatic assays consisting of 0.8(M) O-18-enriched phosphate were prepared under pH-buffered conditions. Upon addition of pyrophosphatase, the Raman spectrum of the solution immediately started to shift to higher wavenumbers, indicating the progressive substitution of O-18 in phosphate by O-16 from water. The analytical resultswere quantified by fitting a Voigt function to themeasured Raman spectra that allowed to determine the relative contribution of each phosphate isotopologue in solution over time. Based on the relative contribution of the different phosphate species, the apparent overall oxygen exchange rate could be calculated assuming a first-order kinetic. The progressive formation and disappearance of the different phosphate isotopologues were thenmodelled by applying a consecutive reaction schemewith first-order steps. The results of our experiments show that Raman spectroscopy can be used to study the kinetics of enzymecatalysed oxygen isotope exchange in the phosphate-water system. Copyright (c) 2016 John Wiley & Sons, Ltd.