Peptide amidase

 

Peptide amidase (PAM) from Strenotrophomonas maltophilia (a gram-negative bacterium) catalyses the hydrolysis of the C-terminal amide bond in peptide amides. It is very regio-selective, and those terminal bonds in amino acid side chains are not hydrolysed. PAM belongs to the amidase signature (AS) family, most of which display hydrolase activity. The natural function of periplasmatic PAM is not known.

 

Reference Protein and Structure

Sequence
Q9ZIV5 UniProt IPR023631 (Sequence Homologues) (PDB Homologues)
Biological species
Bradyrhizobium japonicum (Bacteria) Uniprot
PDB
1ocl - THE CRYSTAL STRUCTURE OF MALONAMIDASE E2 COMPLEXED WITH MALONATE FROM BRADYRHIZOBIUM JAPONICUM (2.0 Å) PDBe PDBsum 1ocl
Catalytic CATH Domains
3.90.1300.10 CATHdb (see all for 1ocl)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.-)

N-acylammonia
CHEBI:83628ChEBI
+
water
CHEBI:15377ChEBI
→
monocarboxylic acid anion
CHEBI:35757ChEBI
+
ammonium
CHEBI:28938ChEBI

Enzyme Mechanism

Introduction

The catalytic mechanism of Ser-Ser-Lys catalytic triad contains five sequential steps: (i) nucleophilic attack of serine on the carbonyl group of the substrate, forming the first tetrahedral intermediate, (ii) formation of an acyl–enzyme complex, (ii) release of an ammonia product, (iv) nucleophilic attack of a water molecule forming the second tetrahedral intermediate, and (iv) the release of the product of the reaction, the carboxylic acid.

Catalytic Residues Roles

UniProt PDB* (1ocl)
Lys62 Lys62A Lys62 is a part of the Ser-Ser-Lys catalytic triad, responsible for accepting from Ser131 and transferring back the proton to Ser131. proton acceptor, proton donor
Ser131 Ser131A Ser131 is a part of the Ser-Ser-Lys catalytic triad, responsible for proton transfer among catalytic residues as well as proton transfer to the amide group of the substrate. proton acceptor, proton donor
Arg158 Arg158A Arg158 establishes an ionic interaction with the carboxyl group of the substrate to ensure correct orientation and alignment of the substrate inside the active site. electrostatic stabiliser
Ser155 Ser155A Ser131 is a part of the Ser-Ser-Lys catalytic triad, responsible for nucleophilic attack for the formation of the acyl-enzyme tetrahedral intermediate. nucleofuge, nucleophile, proton acceptor, proton donor
Thr150, Thr152 (main-N), Gly153 (main-N), Gly154 (main-N) Thr150A, Thr152A (main-N), Gly153A (main-N), Gly154A (main-N) Part of the oxyanion hole for the stabilization of the tetrahedral reaction intermediates.
*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, overall reactant used, intermediate formation, intramolecular elimination, overall product formed, bimolecular elimination

References

  1. Cerqueira NMFSA et al. (2017), Phys Chem Chem Phys, 19, 12343-12354. The mechanism of the Ser-(cis)Ser-Lys catalytic triad of peptide amidases. DOI:10.1039/C7CP00277G. PMID:28453015.
  2. Labahn J et al. (2002), J Mol Biol, 322, 1053-1064. An Alternative Mechanism for Amidase Signature Enzymes. DOI:10.1016/s0022-2836(02)00886-0. PMID:12367528.

Step 1. Ser155 is ionized through proton transfer from this amino acid residue to (cis)Ser131. In the same step, the proton bonded to (cis)Ser131 migrates to Lys62.

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Catalytic Residues Roles

Residue Roles
Lys62A proton acceptor
Ser131A proton donor, proton acceptor
Ser155A proton donor

Chemical Components

proton transfer

Step 2. Ser155 performs nucleophilic attacks towards the carbonyl carbon of malonamate substrate, leading to formation of a tetrahedral adduct. The negatively charged carbonyl group of the amide of the substrate is stabilized by hydrogen bonding provided by the NH groups of the oxyanion pocket.

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Catalytic Residues Roles

Residue Roles
Ser155A nucleophile
Thr152A (main-N) electrostatic stabiliser
Gly153A (main-N) electrostatic stabiliser
Gly154A (main-N) electrostatic stabiliser
Arg158A electrostatic stabiliser

Chemical Components

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

Step 3. The amide group of the tetrahedral intermediate is protonated by (cis)Ser131. At the same time, one proton migrates back from the positively charged Lys62 to (cis)Ser131.

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Catalytic Residues Roles

Residue Roles
Gly154A (main-N) electrostatic stabiliser
Gly153A (main-N) electrostatic stabiliser
Thr152A (main-N) electrostatic stabiliser
Arg158A electrostatic stabiliser
Ser131A proton donor

Chemical Components

proton transfer

Step 4. Formation and release of the first product - ammonia.

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Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: intramolecular elimination, overall product formed

Step 5. The hydroxyl group coming from the water molecule becomes attached to carbon C4 of the acyl–enzyme complex. At the same time, Ser131 and Lys62 are subsequently protonated.

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Catalytic Residues Roles

Residue Roles
Thr152A (main-N) electrostatic stabiliser
Gly154A (main-N) electrostatic stabiliser
Lys62A proton acceptor
Ser131A proton donor, proton acceptor
Gly153A (main-N) electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 6. Formation and release of the second product - malonate.

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Catalytic Residues Roles

Residue Roles
Ser155A nucleofuge

Chemical Components

ingold: bimolecular elimination, overall product formed

Step 7. Regeneration of the original catalytic residues: Ser155 is protonated by proton transfer from Ser131, (cis)Ser131 also become protonated and Lys62 returns to its neutral form.

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Catalytic Residues Roles

Residue Roles
Lys62A proton donor
Ser131A proton donor, proton acceptor
Ser155A proton acceptor

Chemical Components

proton transfer
Download: Image, Marvin File

Step 1. Ser155 is ionized through proton transfer from this amino acid residue to (cis)Ser131. In the same step, the proton bonded to (cis)Ser131 migrates to Lys62.

Step 2. Ser155 performs nucleophilic attacks towards the carbonyl carbon of malonamate substrate, leading to formation of a tetrahedral adduct. The negatively charged carbonyl group of the amide of the substrate is stabilized by hydrogen bonding provided by the NH groups of the oxyanion pocket.

Step 3. The amide group of the tetrahedral intermediate is protonated by (cis)Ser131. At the same time, one proton migrates back from the positively charged Lys62 to (cis)Ser131.

Step 4. Formation and release of the first product - ammonia.

Step 5. The hydroxyl group coming from the water molecule becomes attached to carbon C4 of the acyl–enzyme complex. At the same time, Ser131 and Lys62 are subsequently protonated.

Step 6. Formation and release of the second product - malonate.

Step 7. Regeneration of the original catalytic residues: Ser155 is protonated by proton transfer from Ser131, (cis)Ser131 also become protonated and Lys62 returns to its neutral form.

Products.

Introduction

Ser 226 acts as the primary nucleophile and attacks the amide carbonyl carbon atom. Simultaneously, Ser 202 protonates the substrate carbonyl oxygen, and deprotonates Ser 226. A tetrahedral intermediate is formed. Lys 123 acts to decrease the nucleophilicity of Ser 202, and increase its ability to protonate the carbonyl oxygen. Lys 123 protonates Ser 202 which in turn protonates the amido group of the substrate, creating NH3, a good leaving group. Lys 123 deprotonates Ser 202 which in turn deprotonates the O atom of the substrate, causing the reformation of the carbonyl with the elimination of NH3. Hydrolysis of the enzyme-acyl intermediate is carried out by nucleophilic attack of a water molecule, which is simultaneously deprotonated by the leaving group Ser 226.

Catalytic Residues Roles

UniProt PDB* (1ocl)
Ser202 Ser202(165)A Acts as an acid/base. Donates a proton to the carbonyl oxygen of the substrate at the same time as abstracting a proton from Ser 226. Accepts a proton from Lys 123 while donating a proton to substrate N atom. Accepts a proton from substrate O atom while donating a proton back to Lys 123. proton acceptor, proton relay, electrostatic stabiliser, proton donor
Lys123 Lys123(86)A Acts to decrease the nucleophilicity of Ser 202 by hydrogen bonding to it. Also acts as an acid/base to Ser 202. proton acceptor, electrostatic stabiliser, proton donor
Ser226 Ser226(189)A Acts as the primary nucleophile on the substrate carbonyl. Acts as a base by deprotonating a water molecule as it nucleophilically attacks the substrate carbonyl. nucleofuge, nucleophile, 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

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

References

  1. Labahn J et al. (2002), J Mol Biol, 322, 1053-1064. An Alternative Mechanism for Amidase Signature Enzymes. DOI:10.1016/s0022-2836(02)00886-0. PMID:12367528.
  2. Cerqueira NMFSA et al. (2017), Phys Chem Chem Phys, 19, 12343-12354. The mechanism of the Ser-(cis)Ser-Lys catalytic triad of peptide amidases. DOI:10.1039/C7CP00277G. PMID:28453015.

Step 1. Ser226 as the primary nucleophile attacks the amide carbonyl carbon atom. Simultaneously, the substrate carbonyl group is protonated by Ser202, which in turn deprotonates Ser226.

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Catalytic Residues Roles

Residue Roles
Lys123(86)A electrostatic stabiliser
Ser202(165)A proton acceptor
Ser226(189)A nucleophile
Ser202(165)A proton donor, proton relay
Ser226(189)A proton donor

Chemical Components

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

Step 2. The amido group is protonated by Ser202 to form a plausible leaving group. Ser202 accepts a proton from Lys123.

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Catalytic Residues Roles

Residue Roles
Lys123(86)A electrostatic stabiliser
Lys123(86)A proton donor
Ser202(165)A proton donor, proton acceptor, proton relay

Chemical Components

proton transfer, intermediate formation

Step 3. Lys123 deprotonates Ser202 which allows it to deprotonate the hydroxyl group which initiates elimination resulting in the release of ammonia.

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Catalytic Residues Roles

Residue Roles
Lys123(86)A electrostatic stabiliser
Ser202(165)A proton acceptor
Lys123(86)A proton acceptor
Ser202(165)A proton donor, proton relay

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, intermediate collapse, overall product formed

Step 4. Ser226 abstracts a protonf rom water enabling it to nucleophilically attack the carbon of the acyl-enzyme bond which results in the cleavage of the acyl-enzyme.

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Catalytic Residues Roles

Residue Roles
Lys123(86)A electrostatic stabiliser
Ser202(165)A electrostatic stabiliser
Ser226(189)A nucleofuge, proton acceptor

Chemical Components

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

Step 1. Ser226 as the primary nucleophile attacks the amide carbonyl carbon atom. Simultaneously, the substrate carbonyl group is protonated by Ser202, which in turn deprotonates Ser226.

Step 2. The amido group is protonated by Ser202 to form a plausible leaving group. Ser202 accepts a proton from Lys123.

Step 3. Lys123 deprotonates Ser202 which allows it to deprotonate the hydroxyl group which initiates elimination resulting in the release of ammonia.

Step 4. Ser226 abstracts a protonf rom water enabling it to nucleophilically attack the carbon of the acyl-enzyme bond which results in the cleavage of the acyl-enzyme.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, Charity Hornby, Trung Nguyen, Yordanos Abeje