3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring)

 

The branched-chain alpha-keto dehydrogenase complex catalyzes the overall conversion of alpha-keto acids to acyl-CoA and CO2. It is a multienzyme complex composed of 3 functional components: (E1) branched-chain alpha-keto acid decarboxylase or 2-oxoisovalerate dehydrogenase; (E2) lipoamide acyltransferase; (E3) lipoamide dehydrogenase. This entry represents E2, the lipoamide acyltransferase function, which is a thiamine diphposphate dependent reaction.

 

Reference Protein and Structure

Sequences
P12694 UniProt (1.2.4.4)
P21953 UniProt (1.2.4.4) IPR034616 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1dtw - HUMAN BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE (2.7 Å) PDBe PDBsum 1dtw
Catalytic CATH Domains
3.40.50.970 CATHdb (see all for 1dtw)
Cofactors
Thiamine(1+) diphosphate(3-) (1), Magnesium(2+) (1), Potassium(1+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.2.4.4)

3-methyl-2-oxobutanoate
CHEBI:11851ChEBI
+
N(6)-[(R)-lipoyl]-L-lysine residue
CHEBI:83099ChEBI
+
hydron
CHEBI:15378ChEBI
→
carbon dioxide
CHEBI:16526ChEBI
+
N(6)-[(R)-S(8)-isobutyryldihydrolipoyl]-L-lysine residue
CHEBI:83142ChEBI
Alternative enzyme names: 2-oxoisocaproate dehydrogenase, 2-oxoisovalerate (lipoate) dehydrogenase, 3-methyl-2-oxobutanoate dehydrogenase (lipoamide), 3-methyl-2-oxobutanoate:lipoamide oxidoreductase (decarboxylating and acceptor-2-methylpropanoylating), Alpha-keto-alpha-methylvalerate dehydrogenase, Alpha-ketoisocaproate dehydrogenase, Alpha-ketoisocaproic dehydrogenase, Alpha-ketoisocaproic-alpha-keto-alpha-methylvaleric dehydrogenase, Alpha-ketoisovalerate dehydrogenase, Alpha-oxoisocaproate dehydrogenase, BCKDH, BCOAD, Branched chain keto acid dehydrogenase, Branched-chain (-2-oxoacid) dehydrogenase (BCD), Branched-chain 2-keto acid dehydrogenase, Branched-chain 2-oxo acid dehydrogenase, Branched-chain alpha-keto acid dehydrogenase, Branched-chain alpha-oxo acid dehydrogenase, Branched-chain keto acid dehydrogenase, Branched-chain ketoacid dehydrogenase, Dehydrogenase, 2-oxoisovalerate (lipoate), Dehydrogenase, branched chain alpha-keto acid,

Enzyme Mechanism

Introduction

Glu76' abstracts a proton from the N(1)a position of the thiamine diphosphate cofactor, inducing a proton transfer which activates the cofactor. The cofactor anion attacks at the substrate carbonyl, forming a tetrahedral intermediate. The tetrahedral anion intermediate removes the N(1) proton from the imine ring. This results in the deprotonation of Glu76'. His291 induces decarboxylation from the cofactor-substrate intermediate by abstracting a proton. The disulfide bridge of lipoamide E is broken by nucleophilic attack. His291 initiates the collapse of the intermediate with concomitant product formation. The thiamine diphosphate cofactor is regenerated by abstracting a proton from the positively charged imidazole ring of His291.

Catalytic Residues Roles

UniProt PDB* (1dtw)
His336 His291A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator
Ser337 Ser292A Helps stabilise the reactive intermediates formed. hydrogen bond acceptor, hydrogen bond donor
Ser207 (main-C) Ser162A (main-C) The 'V' conformation of thiamine diphosphate, maintained though interactions with Ser162. hydrogen bond acceptor, steric role
Glu121 Glu76A(BA) Acts as a general acid/base in the activation of the thiamine diphosphate cofactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator, steric role
*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, cofactor used, intermediate formation, bimolecular nucleophilic addition, overall reactant used, bimolecular elimination, decarboxylation, overall product formed, intermediate collapse, bimolecular nucleophilic substitution, intermediate terminated, native state of enzyme regenerated, native state of cofactor regenerated

References

  1. Aevarsson A et al. (1999), Nat Struct Biol, 6, 785-792. Crystal structure of 2-oxoisovalerate and dehydrogenase and the architecture of 2-oxo acid dehydrogenase multienzyme complexes. DOI:10.1038/11563. PMID:10426958.
  2. AEvarsson A et al. (2000), Structure, 8, 277-291. Crystal structure of human branched-chain α-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease. DOI:10.1016/s0969-2126(00)00105-2. PMID:10745006.
  3. Hawes JW et al. (1995), J Biol Chem, 270, 31071-31076. Roles of Amino Acid Residues Surrounding Phosphorylation Site 1 of Branched-chain -Ketoacid Dehydrogenase (BCKDH) in Catalysis and Phosphorylation Site Recognition by BCKDH Kinase. DOI:10.1074/jbc.270.52.31071. PMID:8537366.

Step 1. Glu76' abstracts a proton from the N(1)a position of the thiamine diphosphate cofactor, inducing a proton transfer which activates the cofactor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His291A hydrogen bond acceptor
Glu76A(BA) activator, hydrogen bond acceptor, steric role
Ser292A hydrogen bond donor
Ser162A (main-C) hydrogen bond acceptor, steric role
Glu76A(BA) proton acceptor

Chemical Components

proton transfer, cofactor used, intermediate formation

Step 2. The thiamine cofactor polarises the substrate carbonyl, increasing its reactivity towards the carbanion [PMID:10426958]. The cofactor anion attacks at the substrate carbonyl, forming a tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His291A hydrogen bond donor
Glu76A(BA) hydrogen bond donor, steric role
Ser292A hydrogen bond acceptor
Ser162A (main-C) hydrogen bond acceptor, steric role

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation

Step 3. The tetrahedral anion intermediate removes the N(1) proton from the imine ring. This results in the deprotonation of Glu76'.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His291A hydrogen bond donor, hydrogen bond acceptor
Glu76A(BA) activator, steric role, hydrogen bond donor
Ser292A hydrogen bond acceptor, hydrogen bond donor
Ser162A (main-C) hydrogen bond acceptor, steric role
Glu76A(BA) proton donor

Chemical Components

proton transfer, intermediate formation

Step 4. His291 induces decarboxylation from the cofactor-substrate intermediate by abstracting a proton. The presence of a proton acceptor close to the departing carboxyl group lowers the activation barrier associated with decarboxylation [PMID:10426958].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His291A activator, hydrogen bond acceptor, hydrogen bond donor
Glu76A(BA) hydrogen bond acceptor, steric role
Ser292A hydrogen bond acceptor, hydrogen bond donor
Ser162A (main-C) hydrogen bond acceptor, steric role
His291A proton acceptor

Chemical Components

ingold: bimolecular elimination, decarboxylation, overall product formed, intermediate collapse, intermediate formation

Step 5. The disulfide bridge of lipoamide E is broken by nucleophilic attack.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His291A activator, hydrogen bond donor
Glu76A(BA) hydrogen bond acceptor, steric role
Ser292A hydrogen bond acceptor, hydrogen bond donor
Ser162A (main-C) hydrogen bond acceptor, steric role
His291A proton donor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, intermediate formation, overall reactant used

Step 6. His291 initiates the collapse of the intermediate with concomitant product formation.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His291A activator, hydrogen bond acceptor
Glu76A(BA) hydrogen bond acceptor, steric role
Ser292A hydrogen bond acceptor, hydrogen bond donor
Ser162A (main-C) hydrogen bond acceptor, steric role
His291A proton acceptor

Chemical Components

ingold: bimolecular elimination, intermediate collapse, overall product formed, intermediate formation

Step 7. The thiamine diphosphate cofactor is regenerated by abstracting a proton from the positively charged imidazole ring of His291.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His291A activator, hydrogen bond acceptor, hydrogen bond donor
Glu76A(BA) hydrogen bond acceptor, steric role
Ser292A hydrogen bond acceptor, hydrogen bond donor
Ser162A (main-C) hydrogen bond acceptor, steric role
His291A proton donor

Chemical Components

proton transfer, intermediate terminated, native state of enzyme regenerated, native state of cofactor regenerated
Download: Image, Marvin File

Step 1. Glu76' abstracts a proton from the N(1)a position of the thiamine diphosphate cofactor, inducing a proton transfer which activates the cofactor.

Step 2. The thiamine cofactor polarises the substrate carbonyl, increasing its reactivity towards the carbanion [PMID:10426958]. The cofactor anion attacks at the substrate carbonyl, forming a tetrahedral intermediate.

Step 3. The tetrahedral anion intermediate removes the N(1) proton from the imine ring. This results in the deprotonation of Glu76'.

Step 4. His291 induces decarboxylation from the cofactor-substrate intermediate by abstracting a proton. The presence of a proton acceptor close to the departing carboxyl group lowers the activation barrier associated with decarboxylation [PMID:10426958].

Step 5. The disulfide bridge of lipoamide E is broken by nucleophilic attack.

Step 6. His291 initiates the collapse of the intermediate with concomitant product formation.

Step 7. The thiamine diphosphate cofactor is regenerated by abstracting a proton from the positively charged imidazole ring of His291.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday