Pyridoxal Phosphate Site in Glycogen Phosphorylase b

Structure in Native Enzyme and in Three Derivatives with Modified Cofactors

N. G. Oikonomakos, L. N. Johnson, K. R. Acharya, D. I. Stuart, D. Barford, J. Hajdu, K. M. Varvill, N. G. Oikonomakos, A. E. Melpidou, T. Papageorgiou, D. J. Graves, D. Palm

Research output: Contribution to journalArticle

Abstract

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 [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. 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 31P 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 CH2 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. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1. 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 (P-P distance = 1.0 A). 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.

Original languageEnglish
Pages (from-to)8381-8389
Number of pages9
JournalBiochemistry
Volume26
Issue number25
DOIs
Publication statusPublished - 1 Jan 1987

ASJC Scopus subject areas

  • Biochemistry

Cite this

Pyridoxal Phosphate Site in Glycogen Phosphorylase b : Structure in Native Enzyme and in Three Derivatives with Modified Cofactors. / Oikonomakos, N. G.; Johnson, L. N.; Acharya, K. R.; Stuart, D. I.; Barford, D.; Hajdu, J.; Varvill, K. M.; Oikonomakos, N. G.; Melpidou, A. E.; Papageorgiou, T.; Graves, D. J.; Palm, D.

In: Biochemistry, Vol. 26, No. 25, 01.01.1987, p. 8381-8389.

Research output: Contribution to journalArticle

Oikonomakos, NG, Johnson, LN, Acharya, KR, Stuart, DI, Barford, D, Hajdu, J, Varvill, KM, Oikonomakos, NG, Melpidou, AE, Papageorgiou, T, Graves, DJ & Palm, D 1987, 'Pyridoxal Phosphate Site in Glycogen Phosphorylase b: Structure in Native Enzyme and in Three Derivatives with Modified Cofactors', Biochemistry, vol. 26, no. 25, pp. 8381-8389. https://doi.org/10.1021/bi00399a053
Oikonomakos, N. G. ; Johnson, L. N. ; Acharya, K. R. ; Stuart, D. I. ; Barford, D. ; Hajdu, J. ; Varvill, K. M. ; Oikonomakos, N. G. ; Melpidou, A. E. ; Papageorgiou, T. ; Graves, D. J. ; Palm, D. / Pyridoxal Phosphate Site in Glycogen Phosphorylase b : Structure in Native Enzyme and in Three Derivatives with Modified Cofactors. In: Biochemistry. 1987 ; Vol. 26, No. 25. pp. 8381-8389.
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abstract = "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 [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. 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 31P 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 CH2 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. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1. 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 (P-P distance = 1.0 A). 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.",
author = "Oikonomakos, {N. G.} and Johnson, {L. N.} and Acharya, {K. R.} and Stuart, {D. I.} and D. Barford and J. Hajdu and Varvill, {K. M.} and Oikonomakos, {N. G.} and Melpidou, {A. E.} and T. Papageorgiou and Graves, {D. J.} and D. Palm",
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T2 - Structure in Native Enzyme and in Three Derivatives with Modified Cofactors

AU - Oikonomakos, N. G.

AU - Johnson, L. N.

AU - Acharya, K. R.

AU - Stuart, D. I.

AU - Barford, D.

AU - Hajdu, J.

AU - Varvill, K. M.

AU - Oikonomakos, N. G.

AU - Melpidou, A. E.

AU - Papageorgiou, T.

AU - Graves, D. J.

AU - Palm, D.

PY - 1987/1/1

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N2 - 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 [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. 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 31P 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 CH2 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. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1. 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 (P-P distance = 1.0 A). 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.

AB - 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 [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. 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 31P 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 CH2 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. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1. 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 (P-P distance = 1.0 A). 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.

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