Activator anion binding site in pyridoxal phosphorylase b

The binding of phosphite, phosphate, and fluorophosphate in the crystal

Nikos G. Oikonomakos, Spyros E. Zographos, Katerina E. Tsitsanou, Louise N. Johnson, K. Ravi Acharya

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)2416-2428
Number of pages13
JournalProtein Science
Volume5
Issue number12
DOIs
Publication statusPublished - 1 Jan 1996

Keywords

  • Binding
  • Fluorophosphate
  • Phosphate
  • Phosphite
  • Pyridoxal phosphorylase
  • T state

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Activator anion binding site in pyridoxal phosphorylase b : The binding of phosphite, phosphate, and fluorophosphate in the crystal. / Oikonomakos, Nikos G.; Zographos, Spyros E.; Tsitsanou, Katerina E.; Johnson, Louise N.; Acharya, K. Ravi.

In: Protein Science, Vol. 5, No. 12, 01.01.1996, p. 2416-2428.

Research output: Contribution to journalArticle

Oikonomakos, Nikos G. ; Zographos, Spyros E. ; Tsitsanou, Katerina E. ; Johnson, Louise N. ; Acharya, K. Ravi. / Activator anion binding site in pyridoxal phosphorylase b : The binding of phosphite, phosphate, and fluorophosphate in the crystal. In: Protein Science. 1996 ; Vol. 5, No. 12. pp. 2416-2428.
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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.

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