Hexokinase (type I)

 

Hexokinase catalyses the first step of glycolysis, the phosphorylation of glucose by ATP to produce glucose 6-phosphate (G6P). There are four isozymes of hexokinase in vertebrates where in brain tissue and the red blood cell, type I hexokinase regulates glucose metabolism. As well as phosphorylating glucose, it's substrates also include mannose and fructose.

Yeast Hexokinase is smaller than their vertebrate counterparts but also have isozymes (3). Yeast hexokinase PII has significant structural identity to the mammalian form of the enzyme and also shares the same catalytic residues, therefore thought to have a similar mechanism.

 

Reference Protein and Structure

Sequence
P19367 UniProt (2.7.1.1) IPR001312 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1dgk - MUTANT MONOMER OF RECOMBINANT HUMAN HEXOKINASE TYPE I WITH GLUCOSE AND ADP IN THE ACTIVE SITE (2.8 Å) PDBe PDBsum 1dgk
Catalytic CATH Domains
3.30.420.40 CATHdb (see all for 1dgk)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.7.1.1)

hydroxy group
CHEBI:43176ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
→
phosphate group(2-)
CHEBI:68546ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: ATP-dependent hexokinase, Glucose ATP phosphotransferase, Hexokinase (phosphorylating), Hexokinase D, Hexokinase type IV, Hexokinase type IV glucokinase, Hexokinase type I, Hexokinase type II, Hexokinase type III, Hexokinase type IV (glucokinase),

Enzyme Mechanism

Introduction

The 6-hydroxyl group of Glucose is 3A from the gamma-phosphorus atom and oriented for an in-line displacement reaction. Asp657 is a catalytic base. It abstracts a proton from the 6-hydroxyl group, activating the oxygen which then attacks the gamma-phosphorous. This leads to lysis of the phosphodiester bond forming G6P and ADP. The transition state is stabilised by hydrogen bonding of Ser603 to glucose. Arg539 and the magnesium ion stabilise the developing negative charge on the transition state in the reacton. Asp532, Arg539 and Asp657 all position the magnesium ion via water molecules.

In a recent crystal structure, it is suggested that the pKa of Asp657 is too low to abstract a proton, and that proton transfer is seen to transfer at a later stage on phosphate bond formation with the hydroxyl. However, this is still difficult to show as a different mechanism to the original proposal.

Catalytic Residues Roles

UniProt PDB* (1dgk)
Arg539 Arg539N(A) Alongside the Mg2+ ion present, it serves to stabilise the negatively charged transition state. Also interacts with the magnesium ion via a coordinated water molecule. electrostatic stabiliser, polar interaction
Ser603 Ser603N(A) By analogy with yeast hexokinae PII, Ser603 stabilises the transition state by binding to the 3OH of glucose when it is making a nucleophilic attack on the gamma phosphate electrostatic stabiliser, polar interaction
Asp657 Asp657N(A) Acts as a general base in the reaction, potentially activating oxygen by abstraction of a proton from the 6-hydroxyl position. Additionally interacts with the magnesium ion via a coordinated water molecule. proton acceptor, proton donor
*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

overall product formed, overall reactant used, bimolecular nucleophilic substitution, proton transfer, native state of enzyme regenerated, inferred reaction step

References

  1. Nishimasu H et al. (2007), J Biol Chem, 282, 9923-9931. Crystal structures of an ATP-dependent hexokinase with broad substrate specificity from the hyperthermophilic archaeon Sulfolobus tokodaii. DOI:10.1074/jbc.M610678200. PMID:17229727.
  2. Lewis BE et al. (2003), J Am Chem Soc, 125, 4672-4673. Glucose Binding Isotope Effects in the Ternary Complex of Brain Hexokinase Demonstrate Partial Relief of Ground-State Destabilization. DOI:10.1021/ja029852k. PMID:12696861.
  3. Valiev M et al. (2003), J Am Chem Soc, 125, 9926-9927. The role of the putative catalytic base in the phosphoryl transfer reaction in a protein kinase: first-principles calculations. DOI:10.1021/ja029618u. PMID:12914447.
  4. Aleshin AE et al. (2000), J Mol Biol, 296, 1001-1015. Crystal structures of mutant monomeric hexokinase I reveal multiple ADP binding sites and conformational changes relevant to allosteric regulation. DOI:10.1006/jmbi.1999.3494. PMID:10686099.
  5. Kuser PR et al. (2000), J Biol Chem, 275, 20814-20821. The High Resolution Crystal Structure of Yeast Hexokinase PII with the Correct Primary Sequence Provides New Insights into Its Mechanism of Action. DOI:10.1074/jbc.m910412199. PMID:10749890.
  6. Arora KK et al. (1991), J Biol Chem, 266, 5359-5362. Glucose phosphorylation. Site-directed mutations which impair the catalytic function of hexokinase. PMID:2005085.

Step 1. Asp657 deprotonates the 6'OH on the sugar, activating it for nucleophilic attack on the gamma phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg539N(A) electrostatic stabiliser
Ser603N(A) electrostatic stabiliser, polar interaction
Arg539N(A) polar interaction
Asp657N(A) proton acceptor

Chemical Components

overall product formed, overall reactant used, ingold: bimolecular nucleophilic substitution, proton transfer

Step 2. Deprotonation of Asp657 to regenerate active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser603N(A) polar interaction
Arg539N(A) polar interaction
Asp657N(A) proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Asp657 deprotonates the 6'OH on the sugar, activating it for nucleophilic attack on the gamma phosphate.

Step 2. Deprotonation of Asp657 to regenerate active site.

Products.

Contributors

Gemma L. Holliday, Morwenna Hall, Peter Sarkies