Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli BL21-CodonPlus | Pyrococcus furiosus |
Protein Variants | Comment | Organism |
---|---|---|
D132A | the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. The heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin | Pyrococcus furiosus |
D132A/D136A | the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme | Pyrococcus furiosus |
D132A/N134A | the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. In the presence of EGTA, the protein quantity of very unstable variants enzyme does not decrease as much as that of the wild-type enzyme following heat treatment at 95°C | Pyrococcus furiosus |
D132A/N134A/D136A | the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme | Pyrococcus furiosus |
D132N/D136N | the heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin | Pyrococcus furiosus |
D136A | the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. The heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin | Pyrococcus furiosus |
D138A/Q139A/E140A/D141A | the mutant enzyme displays heat resistance similar to that of the WT under nonchelating conditions but exhibits decreased residual activity compared to the wild-type enzyme following incubation with EGTA at 95°C. These results suggest that the residues downstream of motif 1 also contribute to calcium binding at the Ca3 site | Pyrococcus furiosus |
D161A | maturation defective variant | Pyrococcus furiosus |
D161A/D163A | the variant proform is capable of maturation, but the yield of mature enzyme is very low | Pyrococcus furiosus |
D161A/D163A/D165A | maturation defective variant | Pyrococcus furiosus |
D161A/D163A/D165A/S441A | the D161A/D163A/D165A/S441A proform degrades more than the the S441A proform at 95°C, confirming that the replacement of Asp161, Asp163, and Asp165 with Ala affects structural stability of the proform and makes it more susceptible to thermogenic hydrolysis | Pyrococcus furiosus |
D161A/D165A | maturation defective variant | Pyrococcus furiosus |
D161N/D163N/D165N | the variant proform is capable of maturation, but the yield of mature enzyme is very low. Heat inactivation profile at 95° is similar to wild-type enzyme, either in the absence or presence of EGTA. Similar activity levels as cpompared to wild-type enzyme | Pyrococcus furiosus |
D163A | the variant proform is capable of maturation, but the yield of mature enzyme is very low. Heat inactivation profile at 95° is similar to wild-type enzyme, either in the absence or presence of EGTA. Slightly lower activity levels as cpompared to wild-type enzyme | Pyrococcus furiosus |
D163A/D165A | the variant proform is capable of maturation, but the yield of mature enzyme is very low | Pyrococcus furiosus |
D165A | the variant proform is capable of maturation, but the yield of mature enzyme is very low | Pyrococcus furiosus |
D167A/D170A/E171A | maturation defective variant | Pyrococcus furiosus |
D818N/D820N | half-life at 95°C is 18 h compared to 12 h for wild-type enzyme | Pyrococcus furiosus |
F135R | the thermostability of the variant does not change under nonchelating conditions compared to that of the wild-type. The variant shows a lower level of residual activity than the wild-type enzyme following incubation with EGTA at 95°C. This result implies that a positively charged Arg residue in Dx[DN]xDG motif 1 affects calcium binding at the Ca3 site | Pyrococcus furiosus |
N134A | the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. The heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin | Pyrococcus furiosus |
N134A/D136A | the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. In the presence of EGTA, the protein quantity of very unstable variants enzyme does not decrease as much as that of the wild-type enzyme following heat treatment at 95°C | Pyrococcus furiosus |
R249E | the mutation within insert IS29 improves enzyme thermostability and resistance to salt-induced autocleavage. This is likely due to removing unfavorable electrostatic interactions involving Arg249. Half-life at 95°C is 18 h compared to 12 h for wild-type enzyme | Pyrococcus furiosus |
R249E/D818N/D820N | the mutant enzyme is both resistant to salt induced autocleavage and exhibits a remarkably long half-life of approximately 36 h at 95°C. Half-life at 95°C is 36 h compared to 12 h for wild-type enzyme | Pyrococcus furiosus |
General Stability | Organism |
---|---|
four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin-like proteases is a promising strategy for modulating global structure stability and enzymatic activity of this class of protease | Pyrococcus furiosus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.27 | - |
succinyl-AAPK-4-nitroanilide | 90°C, pH 7.5 | Pyrococcus furiosus |
Organic Solvent | Comment | Organism |
---|---|---|
SDS | mature insert-deletion variants are characterized and show that insert IS29 and insert IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites. One within insert IS29 and the other in insert IS27. Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation | Pyrococcus furiosus |
urea | mature insert-deletion variants are characterized and show that insert IS29 and insert IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites. One within insert IS29 and the other in insert IS27. Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation | Pyrococcus furiosus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pyrococcus furiosus | - |
- |
- |
Purification (Comment) | Organism |
---|---|
- |
Pyrococcus furiosus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
succinyl-AAPK-4-nitroanilide + H2O | - |
Pyrococcus furiosus | succinyl-AAPK + 4-nitroaniline | - |
? |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
additional information | - |
extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments | Pyrococcus furiosus |
95 | - |
half-life: 12 h (wild-type enzyme), 18 h (mutant enzymes R249E and D818N/D820N), 36 h (R249E/D818N/D820N) | Pyrococcus furiosus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1726 | - |
succinyl-AAPK-4-nitroanilide | 90°C, pH 7.5 | Pyrococcus furiosus |