Location of pyridoxal phosphate in glycogen phosphorylase a.

Sygusch, J.; Madsen, Neil B.; Kasvinsky, Peter J.; Fletterick, Robert J.

doi:10.1073/pnas.74.11.4757

Cited by 65 publications

(28 citation statements)

References 26 publications

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“…On the other hand, we see a clear structural difference between AATase and the exceptional pyridoxal-P-dependent enzyme glycogen phosphorylase (EC 2.4.1.1), in which the pyridoxyl aldehyde group does not play a catalytic role (39). In phosphorylase (40,41), pyridoxal-P is attached to the helix linking strands D and E of a six-stranded (C, B, A, D, E, F) a/3 structure that resembles the dinucleotide-binding fold (37). The pyridoxal-P domains can be aligned by the topological equivalence (30) of fragment (f, e, d, b, c) in AATase and fragment (F, E, D, B, C) in phosphorylase.…”

Section: H H H H H H H H H H H H H H H H H H H H H H H H H H H mentioning

confidence: 97%

Three-dimensional structure of a pyridoxal-phosphate-dependent enzyme, mitochondrial aspartate aminotransferase.

Ford¹,

Eichele²,

Jansonius³

1980

Proc. Natl. Acad. Sci. U.S.A.

245

139

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X-ray diffraction studies to 2.8A resolution have yielded the three-dimensional structure of mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), an isologous a2 dimer (M, = 2 X 45,000) The subunits are rich in secondary structure and contain two domains, one of which anchors the coenzyme, pyridoxal 5'-phosphate. Each active site lies between the subunits and is composed of residues from both of them.Aspartate aminotransferase (AATase; L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) is the most studied of the vitamin B-6-dependent enzymes (1, 2). These enzymes catalyze a wide variety of transformations in amino acid metabolism. AATase effects the reversible transfer of an amino group from L-aspartate or L-glutamate to the a-keto acids a-ketoglutarate and oxalacetate. In the course of the double displacement reaction (3, 4) the coenzyme shuttles between the pyridoxal-P form (bound via an aldimine linkage to the E-amino group of a lysine) and the pyridoxamine-P form. "Syncatalytic" conformational changes occur in the enzyme matrix (5-7). Various coenzyme-substrate intermediates are identified by characteristic absorption and circular dichroism spectra (2, 8). The stereochemistry of pyridoxyl-catalyzed reactions has been probed (9), and a dynamic reaction mecha'nism has been proposed (10).Two homologous, genetically independent isozymes of AATase have been found in animal tissues, one in the cytosol (cAATase), the other in the mitochondrial matrix (mAATase). Both are a2 dimers of about 2 X 400 amino acids. The amino acid sequences of the pig (11-13) and chicken (refs. 14 and 15; U. Hausner, K. J. Wilson, and P. Christen, personal communication) isozymes are known or have been almost completely elucidated. Crystallized AATase offers an exceptional opportunity for the study of interactions amongst protein, coenzyme, and substrates during catalysis. The crystalline enzyme is catalytically competent (16). Single-crystal microspectrophotometric studies on cAATase (17, 18) and mAATase (16,19) have permitted the recognition of several reaction intermediates and the evaluation of some dissociation constants and kinetic parameters (19). Now a detailed picture of the enzyme is emerging from X-ray studies of such crystals. The high-resolution X-ray analyses of cAATase from chicken (20) and pig (21) are near completion. Here we report the three-dimensional structure of chicken mAATase in the internal aldimine form (pyridoxal-5'-P-enzyme) as revealed by a 2.8-A resolution X-ray study. RESULTS Structure determinationChicken mAATase crystallizes in a triclinic unit cell that contains one a2 dimer (22) whose subunits are related by a noncrystallographic dyad (23). Diffraction data to 2.8-A resolution of the native protein and the derivatives listed in Table 1 were collected on a CAD4F diffractometer (Enraf-Nonius, Delft, Netherlands). Previously found heavy atom sites (23) were reevaluated by difference Fourier maps. They were refined by alternate cycles of multiple i...

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Section: H H H H H H H H H H H H H H H H H H H H H H H H H H H mentioning

confidence: 97%

Three-dimensional structure of a pyridoxal-phosphate-dependent enzyme, mitochondrial aspartate aminotransferase.

Ford¹,

Eichele²,

Jansonius³

1980

Proc. Natl. Acad. Sci. U.S.A.

245

139

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“…(iii) All the substrates bind near the cofactor, and the phosphate groups of a-D-glucose 1-phosphate (Glc-1-P) and PLP are located close together (7)(8)(9). The phosphate group of P11P is likely to be involved in the catalytic mechanism.…”

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confidence: 99%

Catalytic mechanism of glycogen phosphorylase: pyridoxal(5')diphospho(1)-alpha-D-glucose as a transition-state analogue.

Takagi¹,

Fukui²,

Shimomura³

1982

Proc. Natl. Acad. Sci. U.S.A.

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Pyridoxal(5')diphospho(1)-a-D-glucose was used to reconstitute glycogen phosphorylase b (1,4-a-D-glucan:orthophosphate a-D-glucosyltransferase, EC 2.4.1.1) from rabbit muscle, replacing the natural pyridoxal 5'-phosphate coenzyme. Incubation ofthe reconstituted enzyme alone resulted in the gradual cleavage ofthe synthetic cofactor to pyridoxal 5'-phosphate, which caused slow reactivation of the enzyme. The addition of maltopentaose or glycogen altered the mode of cleavage; the cofactor was rapidly decomposed to pyridoxal 5'-diphosphate. The radioactive glucose moiety released from pyridoxal(5')diphospho(1)-a-D-['4C]glucose was incorporated into the outer chain of glycogen, forming an a-1,4-glucosidic linkage. These results show that the glucosyl transfer reaction discovered mimics the normal catalysis ofthis enzyme, and they strongly support the catalytic mechanism in which the coenzyme phosphate acts as. a catalyst by direct interaction. with the phosphate of the substrate, forming the pyrophosphate-like transition intermediate.The catalytic mechanism of glycogen phosphorylase (1,4-a-Dglucan:orthophosphate a-D.glucosyltransferase, EC 2.4.1.1) remains unclear despite intensive investigations. However, evidence has been accumulating supporting the catalytic function of pyridoxal 5'-phosphate (PLP) bound to this enzyme: (i) The amino acid sequences around the cofactor-linked residues are highly conserved in phosphorylases from many sources (1-4).(ii) There is a large enough space to allow the binding ofa bulky group adjacent to the phosphate group of the cofactor, which interacts with positive charges (5, 6). (iii) All the substrates bind near the cofactor, and the phosphate groups of a-D-glucose 1-phosphate (Glc-1-P) and PLP are located close together (7)(8)(9). The phosphate group of P11P is likely to be involved in the catalytic mechanism.On the basis of the results of 31P NMR studies, Withers et aL (10) suggested that the phosphorus ofthe constrained dianion of the-coenzyme could be an electrophile which, by direct interaction with the phosphate group of Glc-1-P, would facilitate the breakage ofthe glucosidic linkage. This hypothesis was substantiated by the recent investigation carried out collaboratively by the Edmonton group and ourselves (11). Rabbit muscle phosphorylase b reconstituted with pyridoxal(5')diphospho(1)-a-Dglucose (PLPP-a-Glc) demonstrated no enzyme activity but was slowly reactivated on prolonged incubation. The reactivation was markedly diminished in the presence of maltopentaose or glycogen. The 31P NMR spectrum of PLPP-a-Glc bound to the enzyme showed that the phosphate group was constrained by positive charges. The addition of maltopentaose, a glycogen analogue and an alternative substrate, resulted in the disappearance of this spectrum and its replacement with one characteristic of pyridoxal, 5'-diphosphate (PLPP) bound. to the enzyme. Therefore, glucose was released from the synthetic coenzyme.This paper describes the results of the identification of pyridoxal compounds produc...

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“…cross-linking similar to that of ADP. This is probably due to its binding to the nucleotide site (Sygusch et al, 1977;Weber et al, 1978;Vandenbunder et al, 1978). On the other hand, if combined with AMP, Glc-1-P induces a very different transition ( Figure 3B) which can be the manifestation of the heterotropic interaction between these ligands (Graves & Wang, 1972;Griffiths et al, 1976b).…”

Section: Resultsmentioning

confidence: 99%

Structural changes in glycogen phosphorylase as revealed by cross-linking with bifunctional diimidates: phosphorylase b

Hajdu¹,

Dombrádi²,

G³

et al. 1979

Biochemistry

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Location of pyridoxal phosphate in glycogen phosphorylase a.

Cited by 65 publications

References 26 publications

Three-dimensional structure of a pyridoxal-phosphate-dependent enzyme, mitochondrial aspartate aminotransferase.

Three-dimensional structure of a pyridoxal-phosphate-dependent enzyme, mitochondrial aspartate aminotransferase.

Catalytic mechanism of glycogen phosphorylase: pyridoxal(5')diphospho(1)-alpha-D-glucose as a transition-state analogue.

Structural changes in glycogen phosphorylase as revealed by cross-linking with bifunctional diimidates: phosphorylase b

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