Pyridoxal phosphate site in glycogen phosphorylase b: structure in native enzyme and in three derivatives with modified cofactors

Journal Article · Mon Dec 14 23:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00399a053· OSTI ID:5357640

Oikonomakos, N G; Johnson, L N; Acharya, K R; Stuart, D I; Barford, D; Hajdu, J; Papageorgiou, T; Graves, D J; Palm, D

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), Journal Name: Biochemistry; (United States) Vol. 26:25; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BIOCHEMISTRY
CARBOHYDRATES
CHEMICAL REACTIONS
CHEMISTRY
DERIVATIZATION
ENZYMES
GLYCOGEN
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MAMMALS
MUSCLES
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 31
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
POLYSACCHARIDES
RABBITS
RESONANCE
SACCHARIDES
STABLE ISOTOPES
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
VERTEBRATES