Cloned (Comment) | Organism |
---|---|
class I AC gene, overexpression of wild-type and mutant catalytic domain, residues 2-446, i.e. Cya2-446, in strain BL21(DE3) | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
D114A | site-directed mutagenesis, the almost inactive mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
D116A | site-directed mutagenesis, the almost inactive mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
D300A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
E185A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
E242A | site-directed mutagenesis, the mutant shows reduced Vmax and altered Km compared to the wild-type enzyme | Escherichia coli |
K136A | site-directed mutagenesis, the mutant shows reduced Vmax and altered Km compared to the wild-type enzyme | Escherichia coli |
K253A | site-directed mutagenesis, the mutant shows reduced Vmax and altered Km compared to the wild-type enzyme | Escherichia coli |
K260A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
K264A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
K332A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
R19A | site-directed mutagenesis, the mutant shows reduced Vmax and altered Km compared to the wild-type enzyme | Escherichia coli |
S103A | site-directed mutagenesis, the mutant has a 17fold higher Km for ATP compared to the wild-type enzyme, and the mutation causes a marked reduction of discrimination between ATP- and ADP- or AMP-derived inhibitors | Escherichia coli |
S106A | site-directed mutagenesis, the mutation reduces the mutant activity to 25% of the wild-type enzyme activity, kinetic analysis show a 58% reduction of the Vmax and a doubling of the Km compared to the wild-type enzyme | Escherichia coli |
S113A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
T189A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
W118A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
W200A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
W249A | site-directed mutagenesis, the mutant shows reduced Vmax and altered Km compared to the wild-type enzyme | Escherichia coli |
W374A | site-directed mutagenesis, the mutant shows reduced Vmax and altered Km compared to the wild-type enzyme | Escherichia coli |
Y394A | site-directed mutagenesis, the mutant shows reduced Vmax and altered Km compared to the wild-type enzyme | Escherichia coli |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
2'(3')-O-(N-methylanthraniloyl)-ADP | slight competitive inhibition | Escherichia coli | |
2'(3')-O-(N-methylanthraniloyl)-AMP | slight competitive inhibition | Escherichia coli | |
2'(3')-O-(N-methylanthraniloyl)-ATP | competitive | Escherichia coli | |
additional information | inhibition of mutant catalytic domains by 2'(3')-O-(N-methylanthraniloyl)-modified nucleotides is reduced compared to the wild-type catalytic domain Cya2-446, overview | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.153 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant Y394A, 10 mM Mg2+ | Escherichia coli | |
0.165 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant W374A, 10 mM Mg2+ | Escherichia coli | |
0.168 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant W249A, 10 mM Mg2+ | Escherichia coli | |
0.201 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant K136A, 10 mM Mg2+ | Escherichia coli | |
0.216 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant E242A, 10 mM Mg2+ | Escherichia coli | |
0.259 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant K253A, 10 mM Mg2+ | Escherichia coli | |
0.27 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain, 10 mM Mg2+ | Escherichia coli | |
0.275 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant E185A, 10 mM Mg2+ | Escherichia coli | |
0.284 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant K260A, 10 mM Mg2+ | Escherichia coli | |
0.33 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant S113A, 10 mM Mg2+ | Escherichia coli | |
0.398 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant T189A, 10 mM Mg2+ | Escherichia coli | |
0.46 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant R19A, 10 mM Mg2+ | Escherichia coli | |
0.533 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant S106A, 10 mM Mg2+ | Escherichia coli | |
0.682 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant K332A, 10 mM Mg2+ | Escherichia coli | |
4.7 | - |
ATP | pH 8.0, 37°C, recombinant catalytic domain mutant S103A, 10 mM Mg2+ | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | the class I enzyme shows a requirement for free metal ions in addition to the MgATP2- complex, operating with a two-metal-ion mechanism in analogy to class II and calss III enzymes. The native enzyme shows very little activity when the concentration of Mg2+ is much lower than that of ATP, and activity rises strongly when the concentration of Mg2+ exceeds that of ATP | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP | Escherichia coli | - |
3',5'-cyclic-AMP + diphosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P00936 | class I AC gene | - |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant catalytic domain Cya2-446 from strain BL21(DE3) by nickel affinity chromatography | Escherichia coli |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
the specific activity of the catalytic domain fragment Cya2-446 and of the holoenzyme is independent of the protein concentration | Escherichia coli |
0.281 | - |
purified recombinant catalytic domain mutant S106A, 10 mM Mg2+ | Escherichia coli |
0.396 | - |
purified recombinant catalytic domain mutant K136A, 10 mM Mg2+ | Escherichia coli |
0.48 | - |
purified recombinant catalytic domain mutant W374A, 10 mM Mg2+ | Escherichia coli |
0.513 | - |
purified recombinant catalytic domain mutant Y394A, 10 mM Mg2+ | Escherichia coli |
0.526 | - |
purified recombinant catalytic domain mutant K253A, 10 mM Mg2+ | Escherichia coli |
0.614 | - |
purified recombinant catalytic domain mutant R19A, 10 mM Mg2+ | Escherichia coli |
0.665 | - |
purified recombinant catalytic domain, 10 mM Mg2+ | Escherichia coli |
0.752 | - |
purified recombinant catalytic domain mutant E242A, 10 mM Mg2+ | Escherichia coli |
0.778 | - |
purified recombinant catalytic domain mutant W249A, 10 mM Mg2+ | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP | - |
Escherichia coli | 3',5'-cyclic-AMP + diphosphate | - |
? | |
ATP | S103 is important for substrate binding docking to the gamma-phosphate group of ATP | Escherichia coli | 3',5'-cyclic-AMP + diphosphate | - |
? | |
additional information | structure-function relationship, overview | Escherichia coli | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | structure-function relationship, overview | Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
class I adenylate cyclase | - |
Escherichia coli |
Cya | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
45 | - |
native holoenzyme | Escherichia coli |
47 | - |
recombinant catalytic domain Cya2?446 | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
native holoenzyme | Escherichia coli |
8.5 | 9 | recombinant catalytic domain Cya2-446 | Escherichia coli |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.05 | - |
2'(3')-O-(N-methylanthraniloyl)-ATP | pH 8.0, 37°C, versus ATP, wild-type catalytic domain Cya2-446 | Escherichia coli |