TY - JOUR
T1 - Activator anion binding site in pyridoxal phosphorylase b
T2 - The binding of phosphite, phosphate, and fluorophosphate in the crystal
AU - Oikonomakos, Nikos G.
AU - Zographos, Spyros E.
AU - Tsitsanou, Katerina E.
AU - Johnson, Louise N.
AU - Acharya, K. Ravi
PY - 1996/1/1
Y1 - 1996/1/1
N2 - It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5′-phosphate (Chang YC, McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P43212, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 Å resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5′-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 Å). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis.
AB - It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5′-phosphate (Chang YC, McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P43212, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 Å resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5′-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 Å). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis.
KW - Binding
KW - Fluorophosphate
KW - Phosphate
KW - Phosphite
KW - Pyridoxal phosphorylase
KW - T state
UR - http://www.scopus.com/inward/record.url?scp=0030300075&partnerID=8YFLogxK
U2 - 10.1002/pro.5560051204
DO - 10.1002/pro.5560051204
M3 - Article
C2 - 8976550
AN - SCOPUS:0030300075
SN - 0961-8368
VL - 5
SP - 2416
EP - 2428
JO - Protein Science
JF - Protein Science
IS - 12
ER -