Effect of phosphate analogs on the activity of pyridoxal reconstituted glycogen phosphorylase

Parrish, Richard F.; Uhing, Ronald J.; Graves, Donald J.

doi:10.1021/bi00641a011

Cited by 71 publications

(63 citation statements)

References 41 publications

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“…Further chemical evidence to support the involvement of the phosphate moiety was the fi nding by Graves and his colleagues that phosphite would restore the activity of pyridoxal reconstituted phosphorylase b, while pyro phosphate was competitive with both the phosphite activation and the glucose-l-P substrate (32). Since one molecule of pyrophosphate bound tightly to one monomer of the pyridoxal reconstituted enzyme, this sug gested that the phosphate moieties of the coenzyme and substrate are close together, a conclusion verified by the crystal structure (33).…”

Section: Chemical Studiesmentioning

confidence: 99%

The Structures and Related Functions of Phosphorylase a

Fletterick¹,

Madsen²

1980

Annu. Rev. Biochem.

205

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Section: Chemical Studiesmentioning

confidence: 99%

The Structures and Related Functions of Phosphorylase a

Fletterick¹,

Madsen²

1980

Annu. Rev. Biochem.

205

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“…The discovery (Fischer et al, 1958) that the Schiff base linkage could be reduced without substantial loss of activity suggested that the catalytic role of the pyridoxal in phosphorylase is quite different from that in other B6-dependent enzymes. Experiments with coenzyme analogs (Shaltiel et al, 1969;Graves & Wang, 1972;Pfeuffer et al, 1972;Parrish et al, 1977) identified the phosphate group of the PLP as the critical catalytic moiety.…”

mentioning

confidence: 99%

Multiple phosphate positions in the catalytic site of glycogen phosphorylase: Structure of the pyridoxal‐5′‐pyrophosphate coenzyme‐substrate analog

Sprang¹,

Madsen²,

Withers³

1992

Protein Science

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The three-dimensional structure of an R-state conformer of glycogen phosphorylase containing the coenzymesubstrate analog pyridoxal-5'-diphosphate at the catalytic site (PLPP-GPb) has been refined by X-ray crystallography to a resolution of 2.87 A. The molecule comprises four subunits of phosphorylase related by approximate 222 symmetry. Whereas the quaternary structure of R-state PLPP-GPb is similar to that of phosphorylase crystallized in the presence of ammonium sulfate (Barford, D. & Johnson, L.N., 1989, Nature 340, 609-616), the tertiary structures differ in that the two domains of the PLPP-GPb subunits are rotated apart by 5" relative to the T-state conformation. Global differences among the four subunits suggest that the major domains of the phosphorylase subunit are connected by a flexible hinge. The two different positions observed for the terminal phosphate of the PLPP are interpreted as distinct phosphate subsites that may be occupied at different points along the reaction pathway. The structural basis for the unique ability of R-state dimers to form tetramers results from the orientation of subunits with respect to the dyad axis of the dimer. Residues in opposing dimers are in proper registration to form tetramers only in the R-state.

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“…Shimomura and Fukui (5) showed that PLPP binds to apophosphorylase at a rate much higher than that for PLP, indicating a strong binding site for such a pyrophosphate moiety. Parrish et al (16) (17) showed in affinity labeling studies that the E-amino group of Lys-573 was located close to PLP. The results ofchemical modification studies by Dreyfus et al (18) and of inhibition studies by Miller et al.…”

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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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Effect of phosphate analogs on the activity of pyridoxal reconstituted glycogen phosphorylase

Cited by 71 publications

References 41 publications

The Structures and Related Functions of Phosphorylase a

The Structures and Related Functions of Phosphorylase a

Multiple phosphate positions in the catalytic site of glycogen phosphorylase: Structure of the pyridoxal‐5′‐pyrophosphate coenzyme‐substrate analog

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

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