Probes for the conformational transitions of phosphorylase . Effect of ligands studied by proton-relaxation enhancement, and chemical reactivities.

Abstract

1 The properties of rabbit muscle phosphorylase a have been studied by proton relaxation enhancement and chemical reactivity. 2 The response of this enzyme to temperature changes and to binding of ligands (AMP and glucose 1-phosphate) has been observed. 3 There is a single Mn2+-binding site per protomer of phosphorylase a having a dissociation constant, Kd of 135 μM at 20°C. All other sites are at least 10 times weaker. 4 The limiting proton relaxation enhancement factor for phosphorylase a is 18.4. This is not significantly altered by AMP, but changes to 12.3 on saturation with glucose 1-phosphate. 5 The variation in proton relaxation enhancement parameters with temperature shows an inflection at around 15°C, suggesting the occurrence of a conformational change in the enzyme at this temperature. 6 Two groups per protomer react with the specific SH reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, the loss of activity corresponding to the reaction of the slower group. 7 AMP-binding decreases the rate of reaction of the slower SH group, while glucose 1-phosphate increases this rate. That of the faster group is unaffected. 8 These properties and their relationship to the corresponding properties of phosphorylase b reported previously are used to relate phosphorylase a conformations to those of phosphorylase b, and to refine the earlier conclusions about the conformational states observed in muscle phosphorylase.