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Literature summary for 3.4.13.9 extracted from

  • Theriot, C.M.; Du, X.; Tove, S.R.; Grunden, A.M.
    Improving the catalytic activity of hyperthermophilic Pyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures (2010), Appl. Microbiol. Biotechnol., 87, 1715-1726.
    View publication on PubMed
Search for more literature for 3.4.13.9

Cloned(Commentary)

Cloned (Comment) Organism
DNA and amino acid sequence determination and analysis, expression of wild-type and mutant enzymes in Escherichia coli strain JD1 (lambdaDE3) Pyrococcus furiosus

Protein Variants

Protein Variants Comment Organism
G39E site-directed mutagenesis, the mutant shows altered kinetics and temperature profile Pyrococcus furiosus
G39E at 35°C, 70°C and 100°C the mutant exhibits higher Vmax and kcat values than wild-type prolidase for Met-Pro. kcat/Km for Met-Pro is 70% of wild-type value at 35°C, kcat/Km for Met-Pro is 80% of wild-type value at 70°C, kcat/Km for Met-Pro is 1.2fold higher than wild-type value at 100°C. Relative specific activity towards Met-Pro at 100°C is 103% of wild-type activity. Relative specific activity towards Leu-Pro at 100°C is 85% of wild-type activity. Relative specific activity towards Phe-Pro at 100°C is 60% of wild-type activity. Relative specific activity towards Ala-Pro at 100°C is 35% of wild-type activity. Relative specific activity towards Gly-Pro at 100°C is 37% of wild-type activity. Relative specific activity towards Arg-Pro at 100°C is 132% of wild-type activity. Catalytic activity of the mutant enzyme has similar response to changes in pH as wild-type enzyme and shows optimal activity at pH 6.0, although the activity is 60% of wild-type activity Pyrococcus furiosus
additional information the use of Pyrococcus furiosus prolidase for organophosphorus nerve agent decontamination is restricted by the fact that this enzyme displays a narrow functional temperature range. Like many other enzymes isolated from hyperthermophiles, Pyrococcus furiosus prolidase has only 50% activity at 80°C and displays little activity at temperatures below 50°C. Therefore a random-mutated Pyrococcus furiosus prolidase gene library is constructed and screened for production of mutants with increased activity at room temperature while maintaining thermostability. The mutant enzyme shows increased activity over the pH range of 5–7. At pH 5.0, the mutant activity is 1.6fold higher than wild-type activity, and at pH 7.0, it is 1.2fold higher than wild type Pyrococcus furiosus
R19G/G39E/K71E/S229T site-directed mutagenesis, the mutant shows altered kinetics and temperature profile Pyrococcus furiosus
R19G/G39E/K71E/S229T mutant enzyme shows a higher activity than wild-type enzyme over a broad range of temperatures, the thermostability of the mutant enzymes is less compared to wild type. At 35°C, 70°C and 100°C the mutant exhibits higher Vmax and kcat values than wild-type prolidase for Met-Pro. kcat/Km for Met-Pro is 1.2fold higher than wild-type value at 35°C, kcat/Km for Met -Pro is 1.8fold higher than wild-type value at 70°C, kcat/Km for Met-Pro is 2.5fold higher than wild-type value at 100°C. Relative specific activity towards Met-Pro at 100°C is 143% of wild-type activity. Relative specific activity towards Leu-Pro at 100°C is 79% of wild-type activity. Relative specific activity towards Phe-Pro at 100°C is 122% of wild-type activity. Relative specific activity towards Ala-Pro at 100°C is 38% of wild-type activity. Relative specific activity towards Gly-Pro at 100°C is 10% of wild-type activity. Relative specific activity towards Arg-Pro at 100°C is 112% of wild-type activity Pyrococcus furiosus
R19G/K71E/S229 site-directed mutagenesis, the mutant shows altered kinetics and temperature profile Pyrococcus furiosus
R19G/K71E/S229T mutant enzyme shows a higher activity than wild-type enzyme over a broad range of temperatures, the thermostability of the mutant enzymes is less compared to wild type. At 35°C, 70°C and 100°C the mutant exhibits higher Vmax and kcat values than wild-type prolidase for Met-Pro. kcat/Km for Met-Pro is 1.1fold higher than wild-type value at 35°C, kcat/Km for Met -Pro comparable to wild-type value at 70°C, kcat/Km for Met-Pro is 2.4fold higher than wild-type value at 100°C. Relative specific activity towards Met-Pro at 100°C is 137% of wild-type activity. Relative specific activity towards Leu-Pro at 100°C is 169% of wild-type activity. Relative specific activity towards Phe-Pro at 100°C is 97% of wild-type activity. Relative specific activity towards Ala-Pro at 100°C is 95% of wild-type activity. Relative specific activity towards Gly-Pro at 100°C is 47% of wild-type activity. Relative specific activity towards Arg-Pro at 100°C is 101% of wild-type activity. Catalytic activity of the mutant enzyme has similar response to changes in pH as wild-type enzyme and shows optimal activity at pH 7.0, although the activity is 89% of wild-type activity Pyrococcus furiosus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
5.7
-
 Met-Pro pH 7.0, 70°C, recombinant wild-type enzyme Pyrococcus furiosus
5.7
-
 Met-Pro pH 7.0, 70°C, wild-type enzyme Pyrococcus furiosus
6.4
-
 Met-Pro pH 7.0, 35°C, wild-type enzyme Pyrococcus furiosus
6.4
-
 Met-Pro pH 7.0, 35°C, recombinant wild-type enzyme Pyrococcus furiosus
6.8
-
 Met-Pro pH 7.0, 70°C, recombinant mutant G39E Pyrococcus furiosus
6.8
-
 Met-Pro pH 7.0, 70°C, wild-type enzyme G39E Pyrococcus furiosus
7.5
-
 Met-Pro pH 7.0, 70°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
7.5
-
 Met-Pro pH 7.0, 70°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
7.9
-
 Met-Pro pH 7.0, 35°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
7.9
-
 Met-Pro pH 7.0, 35°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
7.95
-
 Met-Pro pH 7.0, 100°C, recombinant wild-type enzyme Pyrococcus furiosus
7.95
-
 Met-Pro pH 7.0, 100°C, wild-type enzyme Pyrococcus furiosus
9.2
-
 Met-Pro pH 7.0, 70°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
9.2
-
 Met-Pro pH 7.0, 70°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
11.1
-
 Met-Pro pH 7.0, 100°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
11.1
-
 Met-Pro pH 7.0, 100°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
11.3
-
 Met-Pro pH 7.0, 35°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
11.3
-
 Met-Pro pH 7.0, 35°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
13
-
 Met-Pro pH 7.0, 35°C, recombinant mutant G39E Pyrococcus furiosus
13
-
 Met-Pro pH 7.0, 35°C, mutant enzyme G39E Pyrococcus furiosus
13.7
-
 Met-Pro pH 7.0, 100°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
13.7
-
 Met-Pro pH 7.0, 100°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
14.5
-
 Met-Pro pH 7.0, 100°C, recombinant mutant G39E Pyrococcus furiosus
14.5
-
 Met-Pro pH 7.0, 100°C, wild-type enzyme G39E Pyrococcus furiosus

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+ activates Pyrococcus furiosus

Organism

Organism UniProt Comment Textmining
Pyrococcus furiosus
-
-
-
Pyrococcus furiosus P81535
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain JD1 (lambdaDE3) by heat treatment at 80°C for 30 min, hydrophobic interaction chromatography, and anion exchange chromatography Pyrococcus furiosus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-nitrophenyl soman + H2O
-
 Pyrococcus furiosus ?
-
 ?
Ala-Pro + H2O
-
 Pyrococcus furiosus Ala + Pro
-
 ?
Arg-Pro + H2O
-
 Pyrococcus furiosus Arg + Pro
-
 ?
Gly-Pro + H2O
-
 Pyrococcus furiosus Gly + Pro
-
 ?
Leu-Pro + H2O
-
 Pyrococcus furiosus Leu + Pro
-
 ?
Met-Pro + H2O
-
 Pyrococcus furiosus Met + Pro
-
 ?
Met-Pro + H2O preferred substrate of wild-type and mutant enzymes Pyrococcus furiosus Met + Pro
-
 ?
Phe-Pro + H2O
-
 Pyrococcus furiosus Phe + Pro
-
 ?
Pro-Ala + H2O
-
 Pyrococcus furiosus Pro + Ala
-
 ?

Synonyms

Synonyms Comment Organism
prolidase
-
 Pyrococcus furiosus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
10 100 activities of wild-type and mutant enzymes at 100°C Pyrococcus furiosus

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
35 100 highest activities of wild-type and mutant enzymes at 100°C Pyrococcus furiosus

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
90
-
 mutants K71E/S229T and R19G/G39E/K71E/S229T loose 50% activity after 4 and 3 h, respectively, whereas mutant G39E does not show this loss of activity until 14 h, and the wild-type enzyme retains 50% activity after 21 h Pyrococcus furiosus
90
-
 R19G/K71E/S229T loses 50% of its activity after 4 h, R19G/G39E/K71E/S229T loses 50% of its activity after 3 h, G39E loses 50% of its activity after 14 h, wild-type enzyme loses 50% of its activity after 21 h Pyrococcus furiosus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
123
-
 Met-Pro pH 7.0, 35°C, wild-type enzyme Pyrococcus furiosus
123
-
 Met-Pro pH 7.0, 35°C, recombinant wild-type enzyme Pyrococcus furiosus
167
-
 Met-Pro pH 7.0, 35°C, recombinant mutant G39E Pyrococcus furiosus
167
-
 Met-Pro pH 7.0, 35°C, mutant enzyme G39E Pyrococcus furiosus
171
-
 Met-Pro pH 7.0, 35°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
171
-
 Met-Pro pH 7.0, 35°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
173
-
 Met-Pro pH 7.0, 35°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
173
-
 Met-Pro pH 7.0, 35°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
767
-
 Met-Pro pH 7.0, 70°C, recombinant wild-type enzyme Pyrococcus furiosus
767
-
 Met-Pro pH 7.0, 70°C, wild-type enzyme Pyrococcus furiosus
1017
-
 Met-Pro pH 7.0, 70°C, recombinant mutant G39E Pyrococcus furiosus
1017
-
 Met-Pro pH 7.0, 70°C, wild-type enzyme G39E Pyrococcus furiosus
1217
-
 Met-Pro pH 7.0, 70°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
1217
-
 Met-Pro pH 7.0, 70°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
2104
-
 Met-Pro pH 7.0, 100°C, recombinant wild-type enzyme Pyrococcus furiosus
2104
-
 Met-Pro pH 7.0, 100°C, wild-type enzyme Pyrococcus furiosus
2416
-
 Met-Pro pH 7.0, 70°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
2416
-
 Met-Pro pH 7.0, 70°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
4594
-
 Met-Pro pH 7.0, 100°C, wild-type enzyme G39E Pyrococcus furiosus
6396
-
 Met-Pro pH 7.0, 100°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
6396
-
 Met-Pro pH 7.0, 100°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
6995
-
 Met-Pro pH 7.0, 100°C, recombinant mutant G39E Pyrococcus furiosus
7481
-
 Met-Pro pH 7.0, 100°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
7481
-
 Met-Pro pH 7.0, 100°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
-
 Pyrococcus furiosus
7
-
 assay at Pyrococcus furiosus

pH Range

pH Minimum pH Maximum Comment Organism
4 10
-
 Pyrococcus furiosus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
13
-
 Met-Pro pH 7.0, 35°C, recombinant mutant G39E Pyrococcus furiosus
13
-
 Met-Pro pH 7.0, 35°C, mutant enzyme G39E Pyrococcus furiosus
15
-
 Met-Pro pH 7.0, 35°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
15
-
 Met-Pro pH 7.0, 35°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
19
-
 Met-Pro pH 7.0, 35°C, recombinant wild-type enzyme Pyrococcus furiosus
19
-
 Met-Pro pH 7.0, 35°C, wild-type enzyme Pyrococcus furiosus
22
-
 Met-Pro pH 7.0, 35°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
22
-
 Met-Pro pH 7.0, 35°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
132
-
 Met-Pro pH 7.0, 70°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
132
-
 Met-Pro pH 7.0, 70°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
135
-
 Met-Pro pH 7.0, 70°C, recombinant wild-type enzyme Pyrococcus furiosus
135
-
 Met-Pro pH 7.0, 70°C, wild-type enzyme Pyrococcus furiosus
150
-
 Met-Pro pH 7.0, 70°C, recombinant mutant G39E Pyrococcus furiosus
150
-
 Met-Pro pH 7.0, 70°C, wild-type enzyme G39E Pyrococcus furiosus
266
-
 Met-Pro pH 7.0, 100°C, recombinant wild-type enzyme Pyrococcus furiosus
266
-
 Met-Pro pH 7.0, 100°C, wild-type enzyme Pyrococcus furiosus
317
-
 Met-Pro pH 7.0, 100°C, recombinant mutant G39E Pyrococcus furiosus
317
-
 Met-Pro pH 7.0, 100°C, wild-type enzyme G39E Pyrococcus furiosus
322
-
 Met-Pro pH 7.0, 70°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
322
-
 Met-Pro pH 7.0, 70°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
467
-
 Met-Pro pH 7.0, 100°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
467
-
 Met-Pro pH 7.0, 100°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
674
-
 Met-Pro pH 7.0, 100°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
674
-
 Met-Pro pH 7.0, 100°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus