Beta-phosphoglucomutase

 

This enzyme catalyses the interconversion of D-glucose 1-phosphate (G1P) and D-glucose 6-phosphate (G6P), forming beta-D-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. It requires Mg(II) and phosphorylation of an aspartate residue at the active site. The enzyme is able to autophosphorylate itself with its substrate beta-D-glucose 1-phosphate. Although this is a slow reaction, only a single turnover is required for activation. Once the phosphorylated enzyme is formed, it generates the reaction intermediate beta-D-glucose 1,6-bisphosphate, which can be used to phosphorylate the enzyme in subsequent cycles [PMID:16784233].

 

Reference Protein and Structure

Sequence
P71447 UniProt (5.4.2.6) IPR010972 (Sequence Homologues) (PDB Homologues)
Biological species
Lactococcus lactis subsp. lactis Il1403 (Bacteria) Uniprot
PDB
1o08 - Structure of Pentavalent Phosphorous Intermediate of an Enzyme Catalyzed Phosphoryl transfer Reaction observed on cocrystallization with Glucose 1-phosphate (1.2 Å) PDBe PDBsum 1o08
Catalytic CATH Domains
3.40.50.1000 CATHdb 1.10.150.240 CATHdb (see all for 1o08)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:5.4.2.6)

beta-D-glucose 1-phosphate(2-)
CHEBI:57684ChEBI
→
beta-D-glucose 6-phosphate(2-)
CHEBI:58247ChEBI
Alternative enzyme names: Beta-pgm (gene name),

Enzyme Mechanism

Introduction

Asp10 initiates the reaction by abstracting a proton from the -CH2-OH group of the substrate sugar, which attacks the phosphate of the phosphorylated Asp8 in a nucleophilic addition. This produces a pentavalent, covalently attached intermediate. The pentavalent, covalently attached intermediate collapses, eliminating Asp8 in an un-phosphorylated state. The bisphosphate intermediate rotates in the active site. Asp8 attacks the C1 phosphate of the intermediate in a nucleophilic addition. This produces a pentavalent, covalently attached intermediate. The intermediate collapses, eliminating the final product and Asp8 in its phosphorylated state.

Catalytic Residues Roles

UniProt PDB* (1o08)
Asp10 (main-N) Asp1010(10)A (main-N) Stabilises the Asp10 side chain. hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Asp8 Asp1008(8)A Forms part of the magnesium binding site. This residue is phosphorylated in the enzyme's ground state. nucleophile, nucleofuge, metal ligand
Asp10 Asp1010(10)A Forms part of the magnesium binding site. Acts as a general acid/base. metal ligand, proton acceptor, proton donor
Leu9 (main-N), Ala115 (main-N), Lys145, Ser114, Thr16, Lys45 Leu1009(9)A (main-N), Ala1115(115)A (main-N), Lys1145(145)A, Ser1114(114)A, Thr1016(16)A, Lys1045(45)A Stabilises the phosphate intermediate. hydrogen bond donor, electrostatic stabiliser
Glu169, Asp170 Glu1169(169)A, Asp1170(170)A Forms part of the magnesium binding site. metal ligand
*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

proton transfer, bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used, unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate collapse, intermediate terminated, native state of enzyme regenerated, overall product formed

References

  1. Lahiri SD et al. (2002), Biochemistry, 41, 8351-8359. Caught in the Act:  The Structure of Phosphorylatedβ-Phosphoglucomutase fromLactococcus lactis†,‡. DOI:10.1021/bi0202373.
  2. Barrozo A et al. (2018), Org Biomol Chem, 16, 2060-2073. Computer simulations of the catalytic mechanism of wild-type and mutant β-phosphoglucomutase. DOI:10.1039/c8ob00312b. PMID:29508879.
  3. Marcos E et al. (2010), Proteins, 78, 2405-2411. Pentacoordinated phosphorus revisited by high-level QM/MM calculations. DOI:10.1002/prot.22758. PMID:20602355.
  4. Dai J et al. (2006), Biochemistry, 45, 7818-7824. Conformational cycling in beta-phosphoglucomutase catalysis: reorientation of the beta-D-glucose 1,6-(Bis)phosphate intermediate. DOI:10.1021/bi060136v. PMID:16784233.
  5. Zhang G et al. (2005), Biochemistry, 44, 9404-9416. Catalytic Cycling in β-Phosphoglucomutase:  A Kinetic and Structural Analysis†,‡. DOI:10.1021/bi050558p. PMID:15996095.
  6. Tremblay LW et al. (2005), J Am Chem Soc, 127, 5298-5299. Chemical Confirmation of a Pentavalent Phosphorane in Complex with β-Phosphoglucomutase. DOI:10.1021/ja0509073. PMID:15826149.
  7. Allen KN et al. (2004), Trends Biochem Sci, 29, 495-503. Phosphoryl group transfer: evolution of a catalytic scaffold. DOI:10.1016/j.tibs.2004.07.008. PMID:15337123.
  8. Lahiri SD et al. (2003), Science, 299, 2067-2071. The Pentacovalent Phosphorus Intermediate of a Phosphoryl Transfer Reaction. DOI:10.1126/science.1082710. PMID:12637673.

Step 1. Asp1010 deprotonates the -CH2-OH group of the substrate sugar, which attacks the phosphate of the phosphorylated Asp1008 in a nucleophilic addition. This produces a pentavalent, covalently attached intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Leu1009(9)A (main-N) hydrogen bond donor, electrostatic stabiliser
Ser1114(114)A hydrogen bond donor, electrostatic stabiliser
Ala1115(115)A (main-N) hydrogen bond donor, electrostatic stabiliser
Asp1010(10)A (main-N) hydrogen bond donor, electrostatic stabiliser
Lys1145(145)A hydrogen bond donor, electrostatic stabiliser
Asp1010(10)A (main-N) hydrogen bond acceptor
Asp1008(8)A metal ligand
Glu1169(169)A metal ligand
Asp1170(170)A metal ligand
Asp1010(10)A metal ligand
Thr1016(16)A electrostatic stabiliser
Lys1045(45)A electrostatic stabiliser
Asp1010(10)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used

Step 2. The pentavalent, covalently attached intermediate collapses, eliminating Asp1008 in an un-phosphorylated state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr1016(16)A electrostatic stabiliser
Lys1045(45)A electrostatic stabiliser
Leu1009(9)A (main-N) hydrogen bond donor, electrostatic stabiliser
Ser1114(114)A hydrogen bond donor, electrostatic stabiliser
Ala1115(115)A (main-N) hydrogen bond donor, electrostatic stabiliser
Asp1010(10)A (main-N) hydrogen bond donor, electrostatic stabiliser
Lys1145(145)A hydrogen bond donor, electrostatic stabiliser
Asp1008(8)A metal ligand
Glu1169(169)A metal ligand
Asp1170(170)A metal ligand
Asp1010(10)A metal ligand
Asp1008(8)A nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate collapse, intermediate formation

Step 3. The bisphosphate intermediate rotates in the active site. Asp1008 attacks the C1 phosphate of the intermediate in a nucleophilic addition. This produces a pentavalent, covalently attached intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp1008(8)A metal ligand
Glu1169(169)A metal ligand
Asp1170(170)A metal ligand
Thr1016(16)A electrostatic stabiliser
Lys1045(45)A electrostatic stabiliser
Ser1114(114)A electrostatic stabiliser, hydrogen bond donor
Ala1115(115)A (main-N) electrostatic stabiliser, hydrogen bond donor
Leu1009(9)A (main-N) electrostatic stabiliser, hydrogen bond donor
Lys1145(145)A electrostatic stabiliser, hydrogen bond donor
Asp1010(10)A (main-N) electrostatic stabiliser, hydrogen bond donor
Asp1010(10)A metal ligand
Asp1008(8)A nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation

Step 4. The pentavalent, covalently attached intermediate collapses, eliminating the final product and Asp1008 in its phosphorylated state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Leu1009(9)A (main-N) electrostatic stabiliser, hydrogen bond donor
Ser1114(114)A electrostatic stabiliser, hydrogen bond donor
Ala1115(115)A (main-N) electrostatic stabiliser, hydrogen bond donor
Asp1010(10)A (main-N) electrostatic stabiliser, hydrogen bond donor
Lys1145(145)A electrostatic stabiliser, hydrogen bond donor
Asp1008(8)A metal ligand
Glu1169(169)A metal ligand
Asp1170(170)A metal ligand
Asp1010(10)A metal ligand
Thr1016(16)A electrostatic stabiliser
Lys1045(45)A electrostatic stabiliser
Asp1010(10)A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, enzyme-substrate complex cleavage, intermediate terminated, intermediate collapse, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. Asp1010 deprotonates the -CH2-OH group of the substrate sugar, which attacks the phosphate of the phosphorylated Asp1008 in a nucleophilic addition. This produces a pentavalent, covalently attached intermediate.

Step 2. The pentavalent, covalently attached intermediate collapses, eliminating Asp1008 in an un-phosphorylated state.

Step 3. The bisphosphate intermediate rotates in the active site. Asp1008 attacks the C1 phosphate of the intermediate in a nucleophilic addition. This produces a pentavalent, covalently attached intermediate.

Step 4. The pentavalent, covalently attached intermediate collapses, eliminating the final product and Asp1008 in its phosphorylated state.

Products.

Contributors

André Minoche, Gemma L. Holliday, Anna Waters, Craig Porter, James Willey