Catalytic site of glycogen phosphorylase: structural changes during activation and mechanistic implications
Withers, S.G.; Madsen, N.B.; Sprang, S.R.; Fletterick, R.J.
Biochemistry 21(21): 5372-5382
The structure of the activated form of [rabbit muscle] glycogen phosphorylase was probed at 2 different levels: in the crystalline state by X-ray crystallography, and in solution by kinetic studies on a modified enzyme using substrate analogs. Addition of a good, nondegradable substrate analog, glucose cyclic 1,2-phosphate (GCP), to crystals of phosphorylase a, previously washed to remove glucose, results in partial activation. Changes occur in the lattice constants, and considerable structural rearrangement is observed upon difference Fourier analysis. The analog is found at essentially the same site as glucose but with a 1 .ANG. translation within the active site. Several protein conformational changes occur in response to this binding and activation, the most prominent being an order .fwdarw. disorder transition affecting a .beta.-hairpin loop (residues 282-286) at the entrance to the active site. This movement creates a substrate phosphate binding pocket within the catalytic site and would increase the accessibility to oligosaccharide substrate. The P atom of GCP is 6.8 .ANG. away from the P of the pyridoxal phosphate (PLP) coenzyme. Kinetic studies with pyridoxal-reconstituted phosphorylase, which is active in the presence of phosphate or a suitable analog as activator, were undertaken to test the hypothetical role of the PLP as a nucleophile in the catalytic mechanism and to elucidate the disposition of the substrate and PLP phosphate groups in the active conformer of the enzyme. The studies with .beta.-D-glucose 1-phosphate are inconsistent with a mechanism in which the PLP phosphate participates in a backside (with respect to the glucosyl C1-O1 bond) attack on the glucosyl C atom to yield a covalent intermediate or to stabilize a glucosyl carbonium ion. The observation of good competitive inhibition by methylenediphosphate, but poor competitive inhibition by ethylene- and propylenediphosphonate, suggests that the phosphate of the coenzyme and that of the substrate may be closer together in the fully activated structure than observed in the X-ray structure of the GCP complex. Possible catalytic mechanisms based on the direct or protein-mediated contact between the substrate and PLP phosphates are discussed in light of these and other recent data.