31P-NMR spectrum of phosphocreatine: Deuterium-induced splitting of the signal

It has been found in experiments with high resolution ³¹P-NMR spectroscopy (200 MHz) that the phosphocreatine peak is splitted into two different peaks in the mixtures of H₂O and D₂O and is single but with different chemical shifts in pure H₂O and D₂O. This phenomenon is explained by substitution of protons of guanidino group in phosphocreatine by deuterium. The effect of splitting disappeared at extreme pH values (>8.5 or <4.0) and at temperatures higher than 45°C due to accelerated proton-deuterium exchange. Creatine kinase added to phosphocreatine solution also lowered its temperature of peaks' collapse by 5°–10°C. A saturation (spin) transfer method was used to show that the phosphoryl group transfer to ADP in creatine kinase active center is slower with deuterium-substituted phosphocreatine than with H-phosphocreatine. The data are taken to show the importance of the proton transfer step in the creatine kinase reaction mechanism and acceleration of phosphocreatine proton-deuterium exchange by creatine kinase.