Pyridoxal phosphate site in glycogen phosphorylase b: structure in native enzyme and in three derivatives with modified cofactors
The detailed environment of the essential cofactor pyridoxal 5'-phosphate in glycogen phosphorylase b, resulting from crystallographic refinement at 1.9-A resolution, is described. The pyridoxal ring is buried in a nonpolar site containing three aromatic rings while the 5'-phosphate group is highly solvated and makes only three direct contacts to the protein. The pyridine nitrogen interacts via a water with protein atoms. The crystal structures of three active derivatives of phosphorylase reconstituted with 5'-deoxypyridoxal 5'-methylenephosphonate (PDMP), 6-fluoropyridoxal 5'-phosphate (6-FPLP), and pyridoxal (PL) in place of the natural cofactor have been determined at 2.5-A resolution. The results for PDMP-phosphorylase show a closer proximity of the phosphonate group to the NZ atom of a lysine (Lys-574) than that observed in the native enzyme, consistent with /sup 31/P NMR studies that have shown a change in ionization state of the phosphonate group compared to the native cofactor phosphate. The replacement of the polar 5'-ester linkage by a CH/sub 2/ group results in a small shift of a water and its hydrogen-bonded tyrosine (Tyr-648). In 6-FPLP-phosphorylase the fluorine is accommodated with no significant change in structure. In PL-phosphorylase co-crystallized with 6.5 mM phosphite and 50 mM glucose, the phosphite anion binds to a site that is close to but distinguishable from the 5'-phosphate site of the coenzyme. In the presence of glucose, phosphite binding provides significant stability to the crystal structure of PL-phosphorylase (T state) through a number of polar interactions. The implications of these results on the role of PLP in phosphorylase are discussed.
- Research Organization:
- Univ. of Oxford, England
- OSTI ID:
- 5357640
- Journal Information:
- Biochemistry; (United States), Vol. 26:25
- Country of Publication:
- United States
- Language:
- English
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550201* - Biochemistry- Tracer Techniques