31P NMR spectra of thiophosphate analogues of guanosine nucleotides : pH dependence of chemical shifts and coupling constants

Abstract

Various enzymes use the guanosine nucleotides GMP, GDP and GTP as natural substrates rather than the more common adenosine nucleotides AMP, ADP and ATP. Hydrolysis of a GTP molecule accompanies, for example, the elongation step of protein synthesis on ribosomes. Some 31P NMR studies on adenosine nucleotides bound to enzymes have been done [l]. The study of the phosphorothioate of AMP as a substitute for the unmodified nucleotide has proved to be useful in exploring the mechanism of glycogen phosphorylase because the difference of the 31P chemical shifts of the nucleotide and the corresponding phosphorothioate facilitated the distinction of the two in the 31P NMR spectrum [2]. This difference in chemical shifts makes the phosphorothioates extremely attractive for structural as well as kinetic studies of enzyme-bound nucleotides by NMR methods, especially in light of the seemingly very similar course of reaction of nucleotides and their phosphorothioates as exemplified for glycerol kinase [31. Besides their biochemical applications, these derivatives merit some interest: analogs of the adenosine nucleotides exhibit a larger change in the chemical shifts of all phosphorus atoms in the nucleotide on secondary protonation of the terminal phosphate, and the values of the coupling constants between the phosphorus atom to which the sulfur is bound and the adjacent phosphorus atoms are increased by a considerable amount in the adenosine nucleotides [4]. To provide a set of data useful for further 31P NMR studies of the interaction of guanosine nucleo-