Conformations of Purine Ribosyl 5′‐Nucleotides Bound to Glycogen Phosphorylase b

The effects of the phosphate ion, glucose 1‐phosphate and glycogen on the binding of several nucleotides to phosphorylase b have been investigated by 1 H and 2 H linewidth measurements and by electron spin resonance. A graphical method is proposed to determine the respective contribution of the strong and weak nucleotide sites of the enzyme to the linewidth of H‐8, H‐2 and H‐1′. The contribution of the strong site to the linewidth is governed by the residence time of nucleotides in the case of AMP. clAMP and IMP ternary complexes with phosphorylase b and HPO 4 2‐ and by the transverse relaxation time in that of the GMP ternary complex. It is shown that in this latter complex the GMP takes an anti conformation instead of a syn conformation in its binary complex with phosphorylase b . This change is related to an enhanced activity of the complex. The reorientation correlation time of binary and ternary complexes estimated from 2 H‐8 linewidth is found of the order of 10 −7 s in agreement with our previous 1 H linewidth measurements. The dependence of the binding of a nucleotide on the concentrations of substrates is studied by the electron spin resonance of N 6 ‐(2,2,6,6‐tetramethyl‐piperidine‐4‐yl‐1‐oxy)‐adenosine 5′‐monophosphate. The proton relaxation induced by the nitroxide group of this compound indicates a distance of the order of 1–2 nm between the two nucleotide strong sites of phosphorylase b .