Glycogen phosphorylase

 

Phophorylases are important allosteric enzymes in the carbohydrate metabolism. Enzymes from different sources differ in their regulatory mechanisms and in their natural substrates. However all known phosphorylases share catalytic and structural properties. For example glycogen phosphorylase catalyses the intracellular degradation of glycogen into glucose-1-phosphate, the first step of glycolysis. It was the first enzyme to be discovered that was controlled by phosphorylation. Other phosphorylases include maltodextrin phosphorylase and starch phosphorylase.

 

Reference Protein and Structure

Sequence
P00489 UniProt (2.4.1.1) IPR011833 (Sequence Homologues) (PDB Homologues)
Biological species
Oryctolagus cuniculus (rabbit) Uniprot
PDB
1gpb - GLYCOGEN PHOSPHORYLASE B: DESCRIPTION OF THE PROTEIN STRUCTURE (1.9 Å) PDBe PDBsum 1gpb
Catalytic CATH Domains
3.40.50.2000 CATHdb (see all for 1gpb)
Cofactors
Pyridoxal 5'-phosphate(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:2.4.1.1)

alpha-maltose
CHEBI:18167ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
→
alpha-D-glucose 1-phosphate(2-)
CHEBI:58601ChEBI
+
alpha-D-glucose
CHEBI:17925ChEBI
Alternative enzyme names: 1,4-alpha-glucan phosphorylase, Alpha-glucan phosphorylase, Amylopectin phosphorylase, Amylophosphorylase, Glucan phosphorylase, Glucosan phosphorylase, Granulose phosphorylase, Maltodextrin phosphorylase, Muscle phosphorylase, Muscle phosphorylase a and b, Myophosphorylase, Polyphosphorylase, Potato phosphorylase, Starch phosphorylase, 1,4-alpha-D-glucan:phosphate alpha-D-glucosyltransferase, Phosphorylase,

Enzyme Mechanism

Introduction

The coenzyme pyridoxal phosphate (PLP) is covalently bound to Lys568 as a Schiff Base. Glycogen substrate binding (shown here as maltose) is followed by a conformational change which brings the substrate close to the coenzyme pyridoxal phosphate (PLP) resulting in the distortion and enhanced electrophilicity of the latter thus labelling the glucosyl phosphate linkage. Bond cleavage then ensues with the coenzyme PLP and its neighbouring basic groups, Gly674, Thr675 and Lys573, essentially sequestering the released inorganic phosphate. The glucosyl carbanion generated may be stabilised as an intermediate by a negatively charged enzymic group, attacked directly in a concerted reaction by the oligosaccharide or trapped by an enzymic group as a glucosyl enzyme intermediate. The latter is the most likely option, the glyucosyl bound via a carboxylate group, Glu671. The glucosyl unit now reacts with the nonreducing end of the oligosaccharide substrate and the reaction proceeds with retention of configuration at the glucose moiety. As a result from this Sn1 reaction, the products glucose-1-phosphate and a n-1 glycogen chain are formed.

Catalytic Residues Roles

UniProt PDB* (1gpb)
Lys681 Lys680A Covalently attached to the PLP cofactor. covalently attached
His378 (main-C), His378 His377A (main-C), His377A Activate and stabilises the sugar ring and oxycarbenium cation. electrostatic stabiliser
Lys569, Arg570, Lys575, Thr677 (main-N) Lys568A, Arg569A, Lys574A, Thr676A (main-N) Stabilise and hold the inorganic phosphate in the correct position electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

unimolecular elimination by the conjugate base, proton transfer, cofactor used, overall reactant used, intermediate formation, overall product formed, proton relay, bimolecular nucleophilic addition, intermediate terminated, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Schwarz A et al. (2005), Biochem J, 387, 437-445. Catalytic mechanism of α-retaining glucosyl transfer byCorynebacterium callunaestarch phosphorylase: the role of histidine-334 examined through kinetic characterization of site-directed mutants. DOI:10.1042/bj20041593. PMID:15535798.
  2. Brás NF et al. (2018), ChemMedChem, 13, 1608-1616. Understanding the Rate-Limiting Step of Glycogenolysis by Using QM/MM Calculations on Human Glycogen Phosphorylase. DOI:10.1002/cmdc.201800218. PMID:29905983.
  3. Buschiazzo A et al. (2004), EMBO J, 23, 3196-3205. Crystal structure of glycogen synthase: homologous enzymes catalyze glycogen synthesis and degradation. DOI:10.1038/sj.emboj.7600324. PMID:15272305.
  4. Geremia S et al. (2002), J Mol Biol, 322, 413-423. Enzymatic Catalysis in Crystals of Escherichia coli Maltodextrin Phosphorylase. DOI:10.1016/s0022-2836(02)00771-4. PMID:12217700.
  5. Oikonomakos NG et al. (2002), Bioorg Med Chem, 10, 1313-1319. The 1.76 A resolution crystal structure of glycogen phosphorylase B complexed with glucose, and CP320626, a potential antidiabetic drug. DOI:10.2210/pdb1h5u/pdb. PMID:11886794.
  6. Livanova NB et al. (2002), Biochemistry (Mosc), 67, 1089-1098. Pyridoxal 5'-phosphate as a catalytic and conformational cofactor of muscle glycogen phosphorylase B. DOI:10.1023/a:1020978825802. PMID:12460107.
  7. Leonidas DD et al. (1992), Protein Sci, 1, 1112-1122. Control of phosphorylasebconformation by a modified cofactor: Crystallographic studies on R-state glycogen phosphorylase reconstituted with pyridoxal 5′-diphosphate. DOI:10.1002/pro.5560010905. PMID:1304390.
  8. Barford D et al. (1991), J Mol Biol, 218, 233-260. Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP. DOI:10.1016/0022-2836(91)90887-c. PMID:1900534.
  9. Tagaya M et al. (1984), J Biol Chem, 259, 4860-4865. Catalytic reaction of glycogen phosphorylase reconstituted with a coenzyme-substrate conjugate. PMID:6425278.

Step 1. The ring oxygen of the substrate initiates an elimination of the alcohol product, which deprotonates a non-covalently bound phosphate, which in turn deprotonates the phosphate of the covalently attached PLP cofactor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys680A covalently attached
His377A (main-C) electrostatic stabiliser
His377A electrostatic stabiliser
Arg569A electrostatic stabiliser
Lys574A electrostatic stabiliser
Thr676A (main-N) electrostatic stabiliser
Lys568A electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, cofactor used, overall reactant used, intermediate formation, overall product formed, proton relay

Step 2. The phosphate of the covalently attached PLP cofactor deprotonates the non-covalently bound phosphate, which in turn attacks the intermediate in a nucleophilic addition that produces the second product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys680A covalently attached
His377A (main-C) electrostatic stabiliser
His377A electrostatic stabiliser
Lys568A electrostatic stabiliser
Arg569A electrostatic stabiliser
Lys574A electrostatic stabiliser
Thr676A (main-N) electrostatic stabiliser

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall product formed, intermediate terminated, native state of cofactor regenerated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The ring oxygen of the substrate initiates an elimination of the alcohol product, which deprotonates a non-covalently bound phosphate, which in turn deprotonates the phosphate of the covalently attached PLP cofactor.

Step 2. The phosphate of the covalently attached PLP cofactor deprotonates the non-covalently bound phosphate, which in turn attacks the intermediate in a nucleophilic addition that produces the second product.

Products.

Contributors

Gemma L. Holliday, Anna Waters, Craig Porter, Morwenna Hall