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Literature summary for 3.5.1.32 extracted from

  • Steele, M.; Marcone, M.; Gyles, C.; Chan, V.L.; Odumeru, J.
    Enzymatic activity of Campylobacter jejuni hippurate hydrolase (2006), Protein Eng. Des. Sel., 19, 17-25.
    View publication on PubMed
Search for more literature for 3.5.1.32

Activating Compound

Activating Compound Comment Organism Structure
EDTA 23.9% activation at 10 mM Campylobacter jejuni

Cloned(Commentary)

Cloned (Comment) Organism
gene hipO, expression of His6-tagged enzyme in Escherichia coli strain M15 Campylobacter jejuni

Protein Variants

Protein Variants Comment Organism
D104N site-directed mutagenesis, inactive mutant Campylobacter jejuni
D76N site-directed mutagenesis, the mutant shows over 12fold reduced activity compared to the wild-type enzyme Campylobacter jejuni
E134Q site-directed mutagenesis, inactive mutant Campylobacter jejuni
E135Q site-directed mutagenesis, inactive mutant Campylobacter jejuni
H161A site-directed mutagenesis, inactive mutant Campylobacter jejuni
H356A site-directed mutagenesis, inactive mutant Campylobacter jejuni

Inhibitors

Inhibitors Comment Organism Structure
1,10-phenanthroline 99% inhibition at 0.5 mM Campylobacter jejuni
2-mercaptoethanol 20% inhibition at 2 mM Campylobacter jejuni
Ag+ 98% inhibition at 2 mM Campylobacter jejuni
Ca2+ 55% inhibition at 2 mM Campylobacter jejuni
Cd2+ 65% inhibition at 2 mM Campylobacter jejuni
Co2+ 70% inhibition at 2 mM Campylobacter jejuni
Cu2+ 78% inhibition at 2 mM Campylobacter jejuni
Fe2+
-
 Campylobacter jejuni
additional information no significant inhibition by E64, PMSF, and pepstatin at 0.5 mM Campylobacter jejuni
Ni2+ 65% inhibition at 2 mM Campylobacter jejuni
Zn2+ 35% inhibition at 2 mM Campylobacter jejuni

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.159
-
 N-benzoylglutamate pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
0.169
-
 N-benzoylalanine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
0.195
-
 N-benzoylphenylalanine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
0.303
-
 N-benzoylmethionine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
0.594
-
 N-benzoylvaline pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
0.65
-
 hippurate pH 7.5, 37°C, native enzyme in cell extract Campylobacter jejuni
0.764
-
 hippurate pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
1.769
-
 4-aminobenzoyl-glutamate pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
2.778
-
 hippurate pH 7.5, 37°C, recombinant mutant D76N Campylobacter jejuni
3.377
-
 N-acetylglycine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
5.45
-
 N-acetyl-L-alanine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni

Metals/Ions

Metals/Ions Comment Organism Structure
Ba2+ 10% activation at 10 mM Campylobacter jejuni
K+ 20% activation at 10 mM Campylobacter jejuni
Mg2+ 20% activation at 10 mM Campylobacter jejuni
Zn2+ 1.98 molecules of zinc per enzyme subunit molecule, metalloenzyme Campylobacter jejuni

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
42400
-
 4 * 42400, recombinant His6-tagged enzyme, SDS-PAGE Campylobacter jejuni
193000
-
 recombinant His6-tagged enzyme, gel filtration Campylobacter jejuni

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
hippurate + H2O Campylobacter jejuni
-
 benzoate + glycine
-
 ?

Organism

Organism UniProt Comment Textmining
Campylobacter jejuni
-
 strain ATCC 43431, gene hipO
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged enzyme from Escherichia coli strain M15 by nickel affinity chromatography Campylobacter jejuni

Reaction

Reaction Comment Organism Reaction ID
hippurate + H2O = benzoate + glycine residues Asp76, Aps104, Glu134, Glu135, His161 and His356 are involved in zinc-binding and enzyme catalysis Campylobacter jejuni
a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
120
-
 purified recombinant wild-type enzyme Campylobacter jejuni

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-aminobenzoyl-glutamate + H2O
-
 Campylobacter jejuni 4-aminobenzoate + glutamate
-
 ?
hippurate + H2O
-
 Campylobacter jejuni benzoate + glycine
-
 ?
hippurate + H2O i.e. N-benzoylglycine Campylobacter jejuni benzoate + glycine
-
 ?
additional information substrate specificity, the enzyme also shows metallocarboxypeptidase activity with carboxypeptidase substrates in vitro, displaying its highest activity against N-benzoyl-linked small aliphatic acids, overview, the enzyme does not hydrolyze typical substrates for aminopeptidases, lysine-p-nitroanilide, leucine-p-nitroanilide, or endopeptidases, N-benzoylarginine-p-nitroanilide, succinylphenylalanine-4-nitroanilide Campylobacter jejuni ?
-
 ?
N-acetylalanine + H2O
-
 Campylobacter jejuni acetate + alanine
-
 ?
N-acetylglycine + H2O
-
 Campylobacter jejuni acetate + glycine
-
 ?
N-benzoylalanine + H2O
-
 Campylobacter jejuni benzoate + alanine
-
 ?
N-benzoylglutamate + H2O
-
 Campylobacter jejuni benzoate + glutamate
-
 ?
N-benzoylmethionine + H2O
-
 Campylobacter jejuni benzoate + methionine
-
 ?
N-benzoylphenylalanine + H2O
-
 Campylobacter jejuni benzoate + phenylalanine
-
 ?
N-benzoylvaline + H2O
-
 Campylobacter jejuni benzoate + valine
-
 ?

Subunits

Subunits Comment Organism
More the enzyme structure shows highly ordered alpha-helix and beta-sheet sequences Campylobacter jejuni
tetramer 4 * 42400, recombinant His6-tagged enzyme, SDS-PAGE Campylobacter jejuni

Synonyms

Synonyms Comment Organism
hippurate hydrolase
-
 Campylobacter jejuni

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
-
 Campylobacter jejuni

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.052 2.1 N-acetylglycine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
0.17
-
 N-benzoylglutamate pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
0.5
-
 N-benzoylvaline pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
1.4
-
 hippurate pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
2 8 N-acetyl-L-alanine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
2.32
-
 hippurate pH 7.5, 37°C, recombinant mutant D76N Campylobacter jejuni
2.6
-
 N-benzoylmethionine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
5.95
-
 N-benzoylphenylalanine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
10.18
-
 N-acetylglycine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
13.33
-
 N-benzoylvaline pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
14.75
-
 4-aminobenzoyl-glutamate pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
22.12
-
 N-acetyl-L-alanine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
29.38
-
 N-benzoylalanine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
48.72
-
 N-benzoylglutamate pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
57.13
-
 N-benzoylmethionine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
81
-
 N-benzoylalanine pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni
98.73
-
 hippurate pH 7.5, 37°C, purified recombinant wild-type enzyme Campylobacter jejuni

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
-
 Campylobacter jejuni