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Literature summary for 3.4.17.18 extracted from

  • Akparov, V.K.; Timofeev, V.I.; Khaliullin, I.G.; ?vedas, V.; Chestukhina, G.G.; Kuranova, I.P.
    Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris (2015), FEBS J., 282, 1214-1224.
    View publication on PubMed
Search for more literature for 3.4.17.18

Crystallization (Commentary)

Crystallization (Comment) Organism
carboxypeptidase T complexes with substrate analogs benzylsuccinic acid and (2-guanidinoethylmercapto)succinic acid by molecular replacement at resolutions of 1.57 A and 1.62 A. The conservative Leu211 and Leu254 residues are structural determinants for recognition of hydrophobic substrates, whereas Asp263 is for recognition of positively charged substrates. The Pro248-Asp258 loop interacting with Leu254 and Tyr255 is responsible for recognition of the substrate's C-terminal residue. Substrate binding at the S10 subsite leads to the ligand-dependent shift of this loop, and Leu254 side chain movement induces the conformation rearrangement of the Glu277 residue crucial for catalysis Thermoactinomyces vulgaris

Protein Variants

Protein Variants Comment Organism
D260N decrease in activity with both substrates benzyloxycarbonyl-Ala-Ala-Leu and benzyloxycarbonyl-Ala-Ala-Arg Thermoactinomyces vulgaris
D263N mutant acquires carboxypeptidase A-like selectivity Thermoactinomyces vulgaris
L211Q mutant acquires carboxypeptidase B-like properties Thermoactinomyces vulgaris
L254S increase in activity with both substrates benzyloxycarbonyl-Ala-Ala-Leu and benzyloxycarbonyl-Ala-Ala-Arg Thermoactinomyces vulgaris
T257D decrease in activity with substrate benzyloxycarbonyl-Ala-Ala-Leu, increase in activity with substrate benzyloxycarbonyl-Ala-Ala-Arg Thermoactinomyces vulgaris
T262G increase in catalytic activity Thermoactinomyces vulgaris

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.04
-
 benzyloxycarbonyl-Ala-Ala-Leu wild-type, pH 7.5, 25°C Thermoactinomyces vulgaris
0.053
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant D260N, pH 7.5, 25°C Thermoactinomyces vulgaris
0.06
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant T262G, pH 7.5, 25°C Thermoactinomyces vulgaris
0.087
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant D263N, pH 7.5, 25°C Thermoactinomyces vulgaris
0.27
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant L254S, pH 7.5, 25°C Thermoactinomyces vulgaris
0.27
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant L254S, pH 7.5, 25°C Thermoactinomyces vulgaris
0.31
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant T257D, pH 7.5, 25°C Thermoactinomyces vulgaris
0.43
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant L211Q, pH 7.5, 25°C Thermoactinomyces vulgaris
0.52
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant L211Q, pH 7.5, 25°C Thermoactinomyces vulgaris
0.59
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant T262G, pH 7.5, 25°C Thermoactinomyces vulgaris
0.67
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant T257D, pH 7.5, 25°C Thermoactinomyces vulgaris
0.69
-
 benzyloxycarbonyl-Ala-Ala-Arg wild-type, pH 7.5, 25°C Thermoactinomyces vulgaris
0.87
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant D260N, pH 7.5, 25°C Thermoactinomyces vulgaris
1.35
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant D263N, pH 7.5, 25°C Thermoactinomyces vulgaris

Organism

Organism UniProt Comment Textmining
Thermoactinomyces vulgaris P29068
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
benzyloxycarbonyl-Ala-Ala-Arg + H2O
-
 Thermoactinomyces vulgaris benzyloxycarbonyl-Ala-Ala + L-Arg
-
 ?
benzyloxycarbonyl-Ala-Ala-Leu + H2O
-
 Thermoactinomyces vulgaris benzyloxycarbonyl-Ala-Ala + L-Leu
-
 ?

Synonyms

Synonyms Comment Organism
CPT
-
 Thermoactinomyces vulgaris

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.6
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant D260N, pH 7.5, 25°C Thermoactinomyces vulgaris
1.1
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant D260N, pH 7.5, 25°C Thermoactinomyces vulgaris
1.1
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant D263N, pH 7.5, 25°C Thermoactinomyces vulgaris
3.6
-
 benzyloxycarbonyl-Ala-Ala-Arg wild-type, pH 7.5, 25°C Thermoactinomyces vulgaris
4.6
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant L211Q, pH 7.5, 25°C Thermoactinomyces vulgaris
4.6
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant L254S, pH 7.5, 25°C Thermoactinomyces vulgaris
4.7
-
 benzyloxycarbonyl-Ala-Ala-Leu wild-type, pH 7.5, 25°C Thermoactinomyces vulgaris
5
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant T257D, pH 7.5, 25°C Thermoactinomyces vulgaris
6.6
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant L254S, pH 7.5, 25°C Thermoactinomyces vulgaris
9.5
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant T257D, pH 7.5, 25°C Thermoactinomyces vulgaris
9.7
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant D263N, pH 7.5, 25°C Thermoactinomyces vulgaris
9.9
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant T262G, pH 7.5, 25°C Thermoactinomyces vulgaris
12.8
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant T262G, pH 7.5, 25°C Thermoactinomyces vulgaris
19
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant L211Q, pH 7.5, 25°C Thermoactinomyces vulgaris

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.68
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant D260N, pH 7.5, 25°C Thermoactinomyces vulgaris
0.84
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant D263N, pH 7.5, 25°C Thermoactinomyces vulgaris
5.3
-
 benzyloxycarbonyl-Ala-Ala-Arg wild-type, pH 7.5, 25°C Thermoactinomyces vulgaris
11
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant L211Q, pH 7.5, 25°C Thermoactinomyces vulgaris
14.1
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant T257D, pH 7.5, 25°C Thermoactinomyces vulgaris
15.9
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant T257D, pH 7.5, 25°C Thermoactinomyces vulgaris
17.4
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant L254S, pH 7.5, 25°C Thermoactinomyces vulgaris
21.5
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant D260N, pH 7.5, 25°C Thermoactinomyces vulgaris
21.7
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant T262G, pH 7.5, 25°C Thermoactinomyces vulgaris
24.3
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant L254S, pH 7.5, 25°C Thermoactinomyces vulgaris
111
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant D263N, pH 7.5, 25°C Thermoactinomyces vulgaris
116
-
 benzyloxycarbonyl-Ala-Ala-Leu wild-type, pH 7.5, 25°C Thermoactinomyces vulgaris
155
-
 benzyloxycarbonyl-Ala-Ala-Leu mutant T262G, pH 7.5, 25°C Thermoactinomyces vulgaris
360
-
 benzyloxycarbonyl-Ala-Ala-Arg mutant L211Q, pH 7.5, 25°C Thermoactinomyces vulgaris