31P nuclear magnetic resonance of bound substrates of arginine kinase reaction: chemical shifts in binary, ternary, quaternary, and transition state analog complexes.

Chemical shifts of fully bound substrates of the arginine kinase reaction were measured in 31P NMR spectra of various binary, ternary, quaternary, and transition state analog complexes. Three of the four substrates of the reaction, namely ATP, ADP, and phosphoarginine contain a total of six phosphate groups and each one can be readily distinguished in the s1P NMR spectra. Binding to the enzyme produces a large chemical shift and line width change only for the P-phosphate resonance of ADP and of Mg. ADP and produces relatively minor effects on ATP, Mg. ATP, and phosphoarginine resonances. The “P-slP spin-spin coupling constants of ADP and ATP are altered by binding to magnesium but are unaffected by binding of the nucleotides or magnesium nucleotides to the enzyme. The structural features of the magnesium chelates of the nucleotides, free and enzyme-bound, revealed by the chemical shifts and ‘j’P-“‘P spin coupling constants are discussed. In particular, it was concluded that both /3and y-phosphates were ligands for Mg2+ in the binary Mg. ATP complex and the ternary E .Mg.ATP complex but chelation of a-phosphate to Mg’+ remains indeterminate in either complex. The protonation of E. Mg. ADP is accompanied by a downfield shift of the resonance position of P-phosphate and exhibits an apparent pK, of 7.5 + 0.3 whereas in the absence of enzyme, protonation causes an upfield shift of the /3phosphate of Mg. ADP with a pK, of 6.0. The pH activity curve of the enzyme in the forward reaction exhibits a pK,, of 7.35. The similarity of the two p& values taken together with the change in direction of the chemical shift of pphosphate upon protonation of the bound versus the free form of Mg. ADP suggests that the state of protonation of an amino acid residue at the active site with a pK,, = 7.5 may determine both the activity of the enzyme and the observed pH dependence of the chemical shift of the P-phosphate of ADP in the E. Mg. ADP complex. 31P chemical shift measurements of ADP in the transition state analog complex