Para-nitrobenzyl esterase

 

Catalyses hydrolysis of several beta-lactam antibiotic PNB esters to the corresponding free acid and PNB alcohol.

 

Reference Protein and Structure

Sequence
P37967 UniProt (3.1.1.-) IPR000997 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus subtilis subsp. subtilis str. 168 (Bacteria) Uniprot
PDB
1qe3 - PNB ESTERASE (1.5 Å) PDBe PDBsum 1qe3
Catalytic CATH Domains
3.40.50.1820 CATHdb (see all for 1qe3)
Click To Show Structure

Enzyme Reaction (EC:3.1.1.-)

water
CHEBI:15377ChEBI
+
N-(4-nitrophenyl)-butyramide
CHEBI:X00684X00684
→
4-nitroaniline
CHEBI:17064ChEBI
+
butyric acid
CHEBI:30772ChEBI

Enzyme Mechanism

Introduction

The enzyme para-Nitrobenzyl (PNB) esterase is a classic Ser-His-Glu triad esterase from the 3.1.1 EC family of proteins. The reaction of para-nitrobenzyl esterase is a classic Ser-His-Asp/Glu triad reaction. His399 is stabilized by Glu310 while deprotonating Ser189. Ser189 then performs a nucleophilic attack on the acyl carbon of the substrate. The nucleophilic attack is followed by the dissociation of 4-nitroanliline from the substrate, leaving the butyraldehyde-protein complex. His399 deprotonates a water molecule which, as a hydroxide binds to the butyraldehyde creating butyric acid. The His399 proton gained by the water molecule in the previous step is given to Ser189, regenerating the active site.

Catalytic Residues Roles

UniProt PDB* (1qe3)
His399 His399A Part of the Ser-His-Glu catalytic triad; activates the serine by acting as a general acid/base. hydrogen bond donor, proton acceptor, proton donor
Glu310 Glu310A Part of the Ser-His-Glu catalytic triad; activates and stabilises the histidine residue. increase basicity, modifies pKa, hydrogen bond acceptor, electrostatic stabiliser
Ser189 Ser189A Part of the Ser-His-Glu catalytic triad; acts as a general acid/base and forms an acyl-enzyme intermediate during the course of the reaction. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, nucleofuge, proton donor, proton acceptor
Gly106 (main-N), Ala107 (main-N), Ala190 (main-N) Gly106A (main-N), Ala107A (main-N), Ala190A (main-N) Gly106(main-N), Ala107(main-N) and Ala190(main-N) form a three point oxyanion hole that stabilizes the acyl intermediates formed in this reaction. This tri-point oxyanion hole is unique to EC 3.1 ester hydrolases. electrostatic stabiliser
*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, overall reactant used, intermediate formation, unimolecular elimination by the conjugate base, intermediate collapse, enzyme-substrate complex cleavage, overall product formed, native state of enzyme regenerated

References

  1. Galmés MÀ et al. (2021), ACS Catal, 8635-8644. Combined Theoretical and Experimental Study to Unravel the Differences in Promiscuous Amidase Activity of Two Nonhomologous Enzymes. DOI:https://doi.org/10.1021/acscatal.1c02150.
  2. Galmés MÀ et al. (2021), ACS Catal, 8635-8644. Combined Theoretical and Experimental Study to Unravel the Differences in Promiscuous Amidase Activity of Two Nonhomologous Enzymes. DOI:https://doi.org/10.1021/acscatal.1c02150.
  3. Legler PM et al. (2014), Front Chem, 2, 46-. Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase. DOI:10.3389/fchem.2014.00046. PMID:25077141.
  4. Ribitsch D et al. (2011), Biotechnol Prog, 27, 951-960. Hydrolysis of polyethyleneterephthalate by p-nitrobenzylesterase from Bacillus subtilis. DOI:10.1002/btpr.610. PMID:21574267.
  5. Spiller B et al. (1999), Proc Natl Acad Sci U S A, 96, 12305-12310. A structural view of evolutionary divergence. PMID:10535917.

Step 1. His399 activates Ser189 to nucleophilically attack the substrate.

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Catalytic Residues Roles

Residue Roles
Glu310A modifies pKa, electrostatic stabiliser
His399A hydrogen bond donor
Glu310A hydrogen bond acceptor
His399A proton acceptor
Ser189A proton donor
Gly106A (main-N) electrostatic stabiliser
Ala107A (main-N) electrostatic stabiliser
Ala190A (main-N) electrostatic stabiliser
Ser189A hydrogen bond donor

Chemical Components

proton transfer

Step 2. Ser189 performs a nucleophilic attack on to the carbonyl carbon creating the acyl-enzyme intermediate.

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Catalytic Residues Roles

Residue Roles
Ser189A nucleophile, hydrogen bond acceptor
His399A hydrogen bond donor
Glu310A modifies pKa, increase basicity
Gly106A (main-N) electrostatic stabiliser
Ala107A (main-N) electrostatic stabiliser
Ala190A (main-N) electrostatic stabiliser
Glu310A electrostatic stabiliser, hydrogen bond acceptor

Chemical Components

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

Step 3. The nitroaniline is cleaved off the rest of the enzyme-acyl complex by recieving a proton from His399. The double bond reforms in the carbonyl oxygen. This step was inferred from the curator.

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Catalytic Residues Roles

Residue Roles
His399A proton donor
Glu310A increase basicity, modifies pKa, electrostatic stabiliser
Ser189A covalently attached
Gly106A (main-N) electrostatic stabiliser
Ala107A (main-N) electrostatic stabiliser
Ala190A (main-N) electrostatic stabiliser
Glu310A hydrogen bond acceptor

Chemical Components

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

Step 4. His399 removes a proton from a water molecule creating a hydroxide that binds to the carbonyl carbon of the substrate.

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Catalytic Residues Roles

Residue Roles
Glu310A electrostatic stabiliser
Gly106A (main-N) electrostatic stabiliser
Ala107A (main-N) electrostatic stabiliser
Ala190A (main-N) electrostatic stabiliser
Ser189A covalently attached
His399A hydrogen bond donor
Glu310A hydrogen bond acceptor
His399A proton acceptor

Chemical Components

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

Step 5. His399 donates a proton to Ser189 collapsing the intermediate. This cleaves the substrate from the enzyme and regenerates the enzymes native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu310A electrostatic stabiliser
Gly106A (main-N) electrostatic stabiliser
Ala107A (main-N) electrostatic stabiliser
Ala190A (main-N) electrostatic stabiliser
Glu310A hydrogen bond acceptor, increase basicity, modifies pKa
Ser189A nucleofuge, proton acceptor
His399A proton donor

Chemical Components

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

Step 1. His399 activates Ser189 to nucleophilically attack the substrate.

Step 2. Ser189 performs a nucleophilic attack on to the carbonyl carbon creating the acyl-enzyme intermediate.

Step 3. The nitroaniline is cleaved off the rest of the enzyme-acyl complex by recieving a proton from His399. The double bond reforms in the carbonyl oxygen. This step was inferred from the curator.

Step 4. His399 removes a proton from a water molecule creating a hydroxide that binds to the carbonyl carbon of the substrate.

Step 5. His399 donates a proton to Ser189 collapsing the intermediate. This cleaves the substrate from the enzyme and regenerates the enzymes native state.

Products.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday, Marko Babić, Antonio Ribeiro