Isotopic ( ¹⁸ O) shift in ³¹ P nuclear magnetic resonance applied to a study of enzyme-catalyzed phosphate—phosphate exchange and phosphate (oxygen)—water exchange reactions

<jats:p> An isotopic shift of the <jats:sup>31</jats:sup> P nuclear magnetic resonance due to <jats:sup>18</jats:sup> O bonded to phosphorus of 0.0206 ppm has been observed in inorganic orthophosphate and adenine nucleotides. Thus, the separation between the resonances of <jats:sup>31</jats:sup> P <jats:sup>18</jats:sup> O <jats:sub>4</jats:sub> and <jats:sup>31</jats:sup> P <jats:sup>16</jats:sup> O <jats:sub>4</jats:sub> at 145.7 MHz is 12 Hz and, in a randomized sample containing ∼50% <jats:sup>18</jats:sup> O, all five <jats:sup>16</jats:sup> O- <jats:sup>18</jats:sup> O species are resolved and separated from each other by 3 Hz. Not only does this yield the <jats:sup>18</jats:sup> O/ <jats:sup>16</jats:sup> O ratio of the phosphate but, more important, the <jats:sup>18</jats:sup> O-labeled phosphate in effect can serve as a double label in following phosphate reactions, for oxygen in all cases and for phosphorus, provided the oxygen does not exchange with solvent water. Thus, it becomes possible to follow labeled phosphorus or labeled oxygen continuously as reactions proceed. Rate studies involving ( <jats:italic>i</jats:italic> ) phosphorus and ( <jats:italic>ii</jats:italic> ) oxygen are illustrated by continuous monitoring of the exchange reactions between ( <jats:italic>i</jats:italic> ) the β phosphate of ADP and inorganic phosphate catalyzed by polynucleotide phosphorylase and ( <jats:italic>ii</jats:italic> ) inorganic orthophosphate and water catalyzed by yeast inorganic pyrophosphatase. In the ADP—P <jats:sub>i</jats:sub> exchange, the P <jats:sub>i</jats:sub> ( <jats:sup>18</jats:sup> O <jats:sub>4</jats:sub> ) yielded an α P( <jats:sup>16</jats:sup> O <jats:sub>3</jats:sub> <jats:sup>18</jats:sup> O) and a β P( <jats:sup>18</jats:sup> O <jats:sub>4</jats:sub> ), proving that bond cleavage occurs between the α P and the α-β bridge oxygen. Among the many additional potential uses of this labeling technique and its spectroscopic observation are: ( <jats:italic>i</jats:italic> ) different labeling of each phosphate group of ATP, ( <jats:italic>ii</jats:italic> ) to follow rate of transfer of <jats:sup>18</jats:sup> O from a nonphosphate compound such as a carboxylic acid to a phosphate compound, and ( <jats:italic>iii</jats:italic> ) to follow the rate of scrambling (for example, of the β-γ bridge oxygen of ATP to nonbridge β P positions) and simultaneously the rate of exchange of the γ P nonbridge oxygens with solvent water in various ATPase reactions. </jats:p>