Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant E151H enzymes in Escherichia coli strain BL21(DE3) | Vibrio proteolyticus |
Crystallization (Comment) | Organism |
---|---|
purified recombinant E151H mutant holoenzyme, 15 mg/ml protein in 10 mM HEPES, pH 7.2, 10 mM KSCN, and 0.4 M NaCl,vapour diffusion mehtod, the precipitation solution contains 100 mM HEPES, pH 7.2, 100 mM KSCN, and 4.5 M NaCl, 2-3 days, X-ray diffraction structure determination and analysis at 1.9 A resolution and room temperature | Vibrio proteolyticus |
Protein Variants | Comment | Organism |
---|---|---|
E151H | site-directed mutagenesis, the mutant enzyme shows a highly reduced reaction rate compared to the wild-type enzyme | Vibrio proteolyticus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | pH-dependence of kinetics, isothermal titration measurement, thermodynamics, overview | Vibrio proteolyticus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Co2+ | can substitute for Zn2+ | Vibrio proteolyticus | |
additional information | detailed metal binding structure analysis, overview, the first metal ion in the dinuclear metal center is in a hexacoordinate/pentacoordinate equilibrium, while the second metal ion is six-coordinate | Vibrio proteolyticus | |
Zn2+ | dinuclear Zn2+ metal center, the binding of Zn2+ to the E151H mutant is much more weak than to the wild-type enzyme | Vibrio proteolyticus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Vibrio proteolyticus | Q01693 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant E151H enzymes from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, dialysis, hydrophobic interaction and anion exchange chromatography | Vibrio proteolyticus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
Release of an N-terminal amino acid, preferentially leucine, but not glutamic or aspartic acids. | active site residue Glu151 is essential for activity acting as a general acid/base during the catalytic reaction, mechanism of peptide hydrolysis | Vibrio proteolyticus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-Leu-4-nitroanilide + H2O | - |
Vibrio proteolyticus | L-Leu + 4-nitroaniline | - |
? |
Synonyms | Comment | Organism |
---|---|---|
AAP | - |
Vibrio proteolyticus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.033 | - |
L-Leu-4-nitroanilide | pH 8.0, recombinant mutant E151H | Vibrio proteolyticus | |
73 | - |
L-Leu-4-nitroanilide | pH 8.0, recombinant wild-type enzyme | Vibrio proteolyticus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | 8.5 | assay at | Vibrio proteolyticus |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
5.5 | 10.8 | pH-profile of mutant E151H | Vibrio proteolyticus |