Probes for the Conformational Transitions of Phosphorylase b Effect of Ligands Studied by Proton Relaxation Enhancement, Fluorescence and Chemical Reactivities

Donald J. Birkett, Raymond A. Dwek, George K. Radda, Rex E. Richards, Andrew G. Salmon

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82 Citations (Scopus)


The effects of temperature and ligands (AMP, IMP and glucose 1‐phosphate) on the properties of rabbit muscle phosphorylase b have been examined by several methods. The non‐covalently bound fluorescent probe 2‐methylanilino naphthalene 6‐sulphonate is shown to detect the dimer to tetramer transition induced by AMP. The aggregation is slow and has a second order rate constant of 3.1×105 M−1 min−1. The interaction of Mn2+ with phosphorylase b is studied by proton relaxation enhancement and electron spin resonance. The limitations of graphical procedures for analysing proton relaxation enhancement data are discussed and better iterative procedures are presented. There are two equivalent and independent binding sites for Mn2+ per phosphorylase b dimer. These sites (Kd= 0.17 mM) have a 30‐fold higher affinity for Mn2+ than those involved in tetramer formation as measured by methylanilino naphthalene sulphonate fluorescence. The reaction of a specific sulphydryl reagent, 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole with phosphorylase b can be separated into sets of one fast and two slowly reacting groups per protomer. Modification of one of the slow groups results in inactivation. The temperature dependences of the proton relaxation rates and of the pattern of – SH reactivity discontinuities at around 13 °C. This has been interpreted as a change in the enzyme conformation. AMP increases the limiting proton relaxation enhancement from 10.9 to 14 and decreases the reactivity with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole of the slow – SH groups. The increase in the limiting proton relaxation enhancement is not due to aggregation but is likely to be a result of a ligand induced conformational chnage. Glucose 1‐phosphate decreases the limiting proton relaxation enhancement from 10.9 to 5.8 and increases the reactivity of one of the slow thiols. On this basis it is argued that the glucose 1‐phosphate induced change is different from that caused by AMP. The limiting proton relaxation enhancement in the presence of both AMP and glucose 1‐phosphate is about 9 suggesting that this represents a fourth conformation. Phosphorylase b modified with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole in the presence of AMP is fully active and the fluorescence of the nitrobenzo‐oxa‐diazole lable is partially quenched when AMP binds. The fluorescence changes show the same AMP concentration dependence as the increase in the proton relaxation enhancement, suggesting that the two probes detect the same transition in the enzyme. A scheme is presented for the activation of phosphorylase b by AMP and involves transitions between four conformations of the enzyme at room temperature. These transitions still occur at low temperature but are superimposed on a different basic form of the enzyme.

Original languageEnglish
Pages (from-to)494-508
Number of pages15
JournalEuropean Journal of Biochemistry
Issue number4
Publication statusPublished - Jun 1971
Externally publishedYes


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