Cloned (Comment) | Organism |
---|---|
overexpression of soluble wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), triple mutant enzyme R147A/R151A/D209A is expressed as insoluble, aggregated protein | Oryctolagus cuniculus |
Protein Variants | Comment | Organism |
---|---|---|
D209A | site-directed mutagenesis, mutant enzyme appears as a mixture of monomeric and dimeric forms, the monomer shows higher thermolability and sensitivity aginst unfolding by 1-anilinonaphthalene-8-sulfonate due to a higher surface area | Oryctolagus cuniculus |
R147A | site-directed mutagenesis, mutant enzyme is a monomer showing higher thermolability and sensitivity aginst unfolding by 1-anilinonaphthalene-8-sulfonate due to a higher surface area, reduced activity and 89% reduced kcat compared to the wild-type enzyme, the mutant enzyme does not follow a random-order rapid-equilibrium mechanism like the wild-type enzyme, but to an ordered mechanism with creatine binding first | Oryctolagus cuniculus |
R147A/R151A | site-directed mutagenesis, double mutant enzyme is a monomer showing higher thermolability and sensitivity aginst unfolding by 1-anilinonaphthalene-8-sulfonate due to a higher surface area, 10fold reduced substrate binding and 40% reduced kcat compared to the wild-type enzyme, the mutant enzyme follows a random-order rapid-equilibrium mechanism like the wild-type enzyme | Oryctolagus cuniculus |
R147A/R151A/D209A | site-directed mutagenesis, the triple mutant enzyme is expressed as insoluble, aggregated protein | Oryctolagus cuniculus |
R151A | site-directed mutagenesis, mutant enzyme is a dimer, reduced activity compared to the wild-type enzyme | Oryctolagus cuniculus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
1-anilinonaphthalene-8-sulfonate | unfolding agent | Oryctolagus cuniculus | |
guanidinium hydrochloride | inactivation mechanism of wild-type and mutant enzymes, overview | Oryctolagus cuniculus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics | Oryctolagus cuniculus |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
cytosol | - |
Oryctolagus cuniculus | 5829 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Oryctolagus cuniculus |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
42000 | - |
recombinant mutant R147A, analytical ultracentrifugation | Oryctolagus cuniculus |
45000 | - |
recombinant mutant R147A/R151A, gel filtration and analytical ultracentrifugation | Oryctolagus cuniculus |
46000 | - |
recombinant mutant R147A, gel filtration | Oryctolagus cuniculus |
47000 | - |
1 * 47000, about, recombinant mutant enzymes D209A, R147A, and R147A/R151A, SDS-PAGE | Oryctolagus cuniculus |
47000 | - |
2 * 47000, about, recombinant wild-type enzyme and recombinant mutant enzymes D209A and R151A, SDS-PAGE | Oryctolagus cuniculus |
67000 | - |
recombinant mutant R151A, analytical ultracentrifugation | Oryctolagus cuniculus |
71000 | - |
recombinant mutant D209A, analytical ultracentrifugation | Oryctolagus cuniculus |
74000 | - |
recombinant mutant D209A, gel filtration | Oryctolagus cuniculus |
81000 | - |
recombinant mutant R151A, gel filtration | Oryctolagus cuniculus |
85000 | - |
recombinant wild-type enzyme, gel filtration | Oryctolagus cuniculus |
86000 | - |
recombinant wild-type enzyme, analytical ultracentrifugation | Oryctolagus cuniculus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + creatine | Oryctolagus cuniculus | - |
ADP + phosphocreatine | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Oryctolagus cuniculus | P00563 | cytosolic isozyme | - |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3) | Oryctolagus cuniculus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
ATP + creatine = ADP + phosphocreatine | random-order rapid-equilibrium mechanism | Oryctolagus cuniculus |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
skeletal muscle | - |
Oryctolagus cuniculus | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + creatine | - |
Oryctolagus cuniculus | ADP + phosphocreatine | - |
r |
Subunits | Comment | Organism |
---|---|---|
dimer | 2 * 47000, about, recombinant wild-type enzyme and recombinant mutant enzymes D209A and R151A, SDS-PAGE | Oryctolagus cuniculus |
monomer | 1 * 47000, about, recombinant mutant enzymes D209A, R147A, and R147A/R151A, SDS-PAGE | Oryctolagus cuniculus |
More | dimerization is no prerequisite for activity but is required for stability and quarternary structure, subunit interface structure and interacting side chains of Glu17, Tyr19, sp53, Gln57, Asp61, Asp209, Arg147, and Arg151, overview | Oryctolagus cuniculus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Oryctolagus cuniculus |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
inactivation of recombinant mutant R147A/R151A | Oryctolagus cuniculus |
40 | 43 | inactivation of recombinant mutant R151A | Oryctolagus cuniculus |
41 | - |
inactivation of recombinant mutant R147A | Oryctolagus cuniculus |
53 | 55 | inactivation of recombinant wild-type enzyme | Oryctolagus cuniculus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
9 | - |
assay at | Oryctolagus cuniculus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ADP | - |
Oryctolagus cuniculus | |
ATP | - |
Oryctolagus cuniculus |