Lactoglutathione lyase

 

Glactoyl-glutathione lyase (or glyoxalase I) is part of the glyoxalase system which catalyses the conversion of acyclic alpha-oxoaldehydes into the corresponding alpha-hydroxyacids. Glyoxalase I catalyses the isomerization of the hemithioacetal (formed spontaneously from alpha-oxoaldehyde and GSH), to S-2-hydroxyacylglutathione (or R) derivatives, therefore decreasing the steady-state concentrations of physiological alpha-oxoaldehydes and associated glycation reactions. Physiological substrates of glyoxalase I are methylglyoxal, glyoxal and other acyclic alpha-oxoaldehydes. This is the first of two steps in the conversion of 2-oxo-aldehydes to the corresponding 2-hydroxycarboxylic acids by way of the glyoxylase system. Methylglyoxal is produced as a by product of the triosephosphate isomerase reaction in glycolysis and, if not removed, is toxic as it reacts readily with with proteins and nucleic acids.

 

Reference Protein and Structure

Sequence
P0AC81 UniProt (4.4.1.5) IPR004361 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1f9z - CRYSTAL STRUCTURE OF THE NI(II)-BOUND GLYOXALASE I FROM ESCHERICHIA COLI (1.5 Å) PDBe PDBsum 1f9z
Catalytic CATH Domains
3.10.180.10 CATHdb (see all for 1f9z)
Cofactors
Nickel(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:4.4.1.5)

methylglyoxal
CHEBI:17158ChEBI
+
glutathione
CHEBI:16856ChEBI
→
(R)-S-lactoylglutathione
CHEBI:15694ChEBI
Alternative enzyme names: Aldoketomutase, Glyoxylase I, Ketone-aldehyde mutase, Methylglyoxalase, Glyoxalase I, (R)-S-lactoylglutathione methylglyoxal-lyase (isomerizing),

Enzyme Mechanism

Introduction

In this mechanism derived for the E. coli enzyme from a crystal structure with bound product, it is postulated that the substrate does not directly bind the metal (as in the human mechanism). The identity of the general acid/base is thus postulated to be the two water molecules that remain bound to the metal and are available to facilitate the proton transfer reactions. Otherwise, the mechanism is broadly similar to the human glyoxalase I. The enzyme only works on the hemithioacetal intermediate which is formed by the spontaneous reaction between methylglyoxal and glutathione. Both the (R) and (S) isomerisation reaction mechanisms form a cis-ene-diol intermediate coordinated directly to the Ni(II) centre. This intermediate then undergoes keto-enol tautomerisation, assisted by Ni(II) bound water molecules in both mechanisms to form the hydroxyacylglutathione product.

Catalytic Residues Roles

UniProt PDB* (1f9z)
Glu122 Glu122A Binds catalytic zinc ion and acts as a general acid/base. hydrogen bond acceptor, metal ligand, proton acceptor, proton donor
Glu56, His74, His5 Glu56B, His74A, His5B Binds catalytic zinc ion. hydrogen bond acceptor, hydrogen bond donor, 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

reaction occurs outside the enzyme, atom stereo change, cofactor used, proton transfer, intermediate formation, assisted keto-enol tautomerisation, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Boer JL et al. (2014), Arch Biochem Biophys, 544, 142-152. Nickel-dependent metalloenzymes. DOI:10.1016/j.abb.2013.09.002. PMID:24036122.
  2. Davidson G et al. (2001), Biochemistry, 40, 4569-4582. An XAS Investigation of Product and Inhibitor Complexes of Ni-Containing GlxI fromEscherichia coli:  Mechanistic Implications†. DOI:10.1021/bi0018537.
  3. He MM et al. (2000), Biochemistry, 39, 8719-8727. Determination of the Structure ofEscherichia coliGlyoxalase I Suggests a Structural Basis for Differential Metal Activation†. DOI:10.1021/bi000856g. PMID:10913283.

Step 1. Spontaneous formation of the lactoylglutathione.

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Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, atom stereo change, cofactor used

Step 2. An active site base deprotonates one of the Ni(II) bound water.

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Catalytic Residues Roles

Residue Roles
Glu56B hydrogen bond acceptor
Glu122A metal ligand, hydrogen bond acceptor
His74A metal ligand
Glu56B metal ligand
His5B metal ligand
Glu122A proton acceptor

Chemical Components

proton transfer, atom stereo change

Step 3. The activated water molecule abstracts a proton from the C3 of the substrate.

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Catalytic Residues Roles

Residue Roles
Glu56B hydrogen bond donor
Glu122A hydrogen bond acceptor
His74A metal ligand
Glu56B metal ligand
Glu122A metal ligand
His5B metal ligand

Chemical Components

proton transfer, intermediate formation, assisted keto-enol tautomerisation

Step 4. The substrate undergoes an assisted keto-enol tautomerisation.

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Catalytic Residues Roles

Residue Roles
Glu56B hydrogen bond acceptor
Glu122A hydrogen bond acceptor
His74A metal ligand
Glu56B metal ligand
Glu122A metal ligand
His5B metal ligand

Chemical Components

proton transfer, assisted keto-enol tautomerisation, atom stereo change, overall product formed

Step 5. The negatively charged oxygen of the product deprotonates a Ni(II) bound water, which in turn deprotonates the Glu122, returning the enzyme to an active starting state.

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Catalytic Residues Roles

Residue Roles
His74A metal ligand
Glu56B metal ligand
Glu122A metal ligand
His5B metal ligand
Glu122A proton donor

Chemical Components

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

Step 1. Spontaneous formation of the lactoylglutathione.

Step 2. An active site base deprotonates one of the Ni(II) bound water.

Step 3. The activated water molecule abstracts a proton from the C3 of the substrate.

Step 4. The substrate undergoes an assisted keto-enol tautomerisation.

Step 5. The negatively charged oxygen of the product deprotonates a Ni(II) bound water, which in turn deprotonates the Glu122, returning the enzyme to an active starting state.

Products.

Introduction

The mechanism employed by the enzyme reflects the stereochemistry of the substrate. Both S and R forms of the hemithioacetal bind in the active site, coordinated to the Ni(II) metal in the place of previously coordinated water molecules. The R enantiomer reaction mechanism differs in that it involves two bases (Glu56 and Glu122) as opposed to only one base in the S case (Glu122). The enzyme only works on the hemithioacetal intermediate which is formed by the spontaneous reaction between methylglyoxal and glutathione. Both reaction mechanisms form a cis-ene-diol intermediate coordinated directly to the Zn centre. This intermediate then undergoes keto-enol tautomerisation, assisted by Glu122 in both mechanisms to form the R-2-hydroxyacylglutathione product. Lactoylgutathione lyase I catalyses the first half of the mechanism to detoxify methyl-glyoxyl, where the product of this reaction is relayed to glyoxylase II (M0157) which catalyses the hydrolysis reaction to form D-lactate and glutathione.

Catalytic Residues Roles

UniProt PDB* (1f9z)
Glu122, Glu56 Glu122A, Glu56B Binds catalytic zinc ion and acts as a general acid/base. hydrogen bond acceptor, metal ligand, proton acceptor, proton donor, proton relay
His74, His5 His74A, His5B Binds catalytic zinc ion. 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

reaction occurs outside the enzyme, atom stereo change, proton transfer, assisted keto-enol tautomerisation, overall reactant used, intermediate formation, overall product formed, intermediate terminated, proton relay, native state of enzyme regenerated, native state of cofactor is not regenerated

References

  1. Boer JL et al. (2014), Arch Biochem Biophys, 544, 142-152. Nickel-dependent metalloenzymes. DOI:10.1016/j.abb.2013.09.002. PMID:24036122.
  2. Suttisansanee U et al. (2011), Semin Cell Dev Biol, 22, 285-292. Bacterial glyoxalase enzymes. DOI:10.1016/j.semcdb.2011.02.004. PMID:21310258.
  3. Clugston SL et al. (2004), Biochem J, 377, 309-316. Investigation of metal binding and activation of Escherichia coli glyoxalase I: kinetic, thermodynamic and mutagenesis studies. DOI:10.1042/bj20030271. PMID:14556652.
  4. Davidson G et al. (2001), Biochemistry, 40, 4569-4582. An XAS Investigation of Product and Inhibitor Complexes of Ni-Containing GlxI fromEscherichia coli:  Mechanistic Implications†. DOI:10.1021/bi0018537.
  5. He MM et al. (2000), Biochemistry, 39, 8719-8727. Determination of the Structure ofEscherichia coliGlyoxalase I Suggests a Structural Basis for Differential Metal Activation†. DOI:10.1021/bi000856g. PMID:10913283.
  6. Cameron AD et al. (1999), Biochemistry, 38, 13480-13490. Reaction Mechanism of Glyoxalase I Explored by an X-ray Crystallographic Analysis of the Human Enzyme in Complex with a Transition State Analogue†. DOI:10.1021/bi990696c. PMID:10521255.

Step 1. Spontaneous formation of the lactoylglutathione.

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Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, atom stereo change

Step 2. Glu56' deprotonates the C3 carbon adjacent to the sulfur atom, resulting in double bond rearrangement and formation of the enol-intermediate.

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Catalytic Residues Roles

Residue Roles
Glu56B hydrogen bond acceptor
Glu122A hydrogen bond acceptor
Glu122A metal ligand
His5B metal ligand
His74A metal ligand
Glu56B metal ligand, proton acceptor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, atom stereo change, overall reactant used, intermediate formation

Step 3. The oxyanion deprotonates Glu56'.

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Catalytic Residues Roles

Residue Roles
Glu56B hydrogen bond donor
Glu122A hydrogen bond acceptor
Glu122A metal ligand
His5B metal ligand
His74A metal ligand
Glu56B metal ligand, proton donor

Chemical Components

proton transfer, intermediate formation

Step 4. Glu122A deprotonates the C3 hydroxy group, causing double bond rearrangement and the protonation of C2 from Glu122A.

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Catalytic Residues Roles

Residue Roles
Glu56B hydrogen bond acceptor
Glu122A hydrogen bond acceptor, proton relay
Glu122A metal ligand
His5B metal ligand
His74A metal ligand
Glu56B metal ligand
Glu122A proton acceptor, proton donor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, atom stereo change, overall product formed, intermediate terminated, proton relay, native state of enzyme regenerated, native state of cofactor is not regenerated
Download: Image, Marvin File

Step 1. Spontaneous formation of the lactoylglutathione.

Step 2. Glu56' deprotonates the C3 carbon adjacent to the sulfur atom, resulting in double bond rearrangement and formation of the enol-intermediate.

Step 3. The oxyanion deprotonates Glu56'.

Step 4. Glu122A deprotonates the C3 hydroxy group, causing double bond rearrangement and the protonation of C2 from Glu122A.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday