Cloned (Comment) | Organism |
---|---|
gene YKR043C, DNA and amino acid sequence determination and analysis, recombinant expression of the N-terminally His6-tagged enzyme, comtaining a tobacco etch virus protease cleavage site, in Escherichia coli strain BL21(DE3) | Saccharomyces cerevisiae |
Crystallization (Comment) | Organism |
---|---|
purified selenomethionine-substituted YK23 in apo-form, hanging drop vapor diffusion method, 22°C, purified YK23, at 29 mg/ml, is mixed with 1.5 mg/ml of trypsin solution in 1 mM HCl and 2 mM CaCl2 in a ratio of 1:10 for the apoenzyme and or 1:20 for the phosphate-bound enzyme, mixing of 0.002 ml of protein solution with 0.002 ml of reservoir solution, containing 0.2 M trilithium citrate, pH 8.1, 16% PEG 3350 and 4% 2-methyl-2,4-pentanediol, 2 days, X-ray diffraction structure determination and analysis at 1.75 A resolution, molecular replacement, modelling | Saccharomyces cerevisiae |
Protein Variants | Comment | Organism |
---|---|---|
E99A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Saccharomyces cerevisiae |
H13A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Saccharomyces cerevisiae |
H178A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Saccharomyces cerevisiae |
H244A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Saccharomyces cerevisiae |
H268A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Saccharomyces cerevisiae |
R181A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Saccharomyces cerevisiae |
R69A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Saccharomyces cerevisiae |
S19A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Saccharomyces cerevisiae |
S65A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Saccharomyces cerevisiae |
W131A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Saccharomyces cerevisiae |
Y24A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Saccharomyces cerevisiae |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
Cd2+ | - |
Saccharomyces cerevisiae | |
Cu2+ | - |
Saccharomyces cerevisiae | |
D-fructose-2,6-bisphosphate | competitive inhibition | Saccharomyces cerevisiae | |
phosphate | - |
Saccharomyces cerevisiae | |
Zn2+ | - |
Saccharomyces cerevisiae |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | with all natural substrates, YK23 shows classical hyperbolic saturation kinetics | Saccharomyces cerevisiae | |
0.2 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, wild-type enzyme | Saccharomyces cerevisiae | |
0.3 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant R69A | Saccharomyces cerevisiae | |
0.5 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant H268A | Saccharomyces cerevisiae | |
0.6 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant Y24A | Saccharomyces cerevisiae | |
0.7 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant S19A | Saccharomyces cerevisiae | |
0.9 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant S65A | Saccharomyces cerevisiae | |
0.9 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant W131A | Saccharomyces cerevisiae | |
1.4 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant R181A | Saccharomyces cerevisiae | |
1.5 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant H178A | Saccharomyces cerevisiae | |
2.3 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant H244A | Saccharomyces cerevisiae |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
additional information | the enzyme is independent of metals, Mg2+, Mn2+, Co2+, Ni2+, Li+, and Ca2+ show no effect on activity | Saccharomyces cerevisiae |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-fructose 1,6-bisphosphate + H2O | Saccharomyces cerevisiae | best substrate | D-fructose 6-phosphate + phosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharomyces cerevisiae | P36136 | gene YKR043C | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-fructose 1,6-bisphosphate + H2O | best substrate | Saccharomyces cerevisiae | D-fructose 6-phosphate + phosphate | - |
? | |
D-fructose 1,6-bisphosphate + H2O | the substrate is bound in its linear, open conformation with the cleavable C1-phosphate positioned deep in the active site | Saccharomyces cerevisiae | D-fructose 6-phosphate + phosphate | - |
? | |
additional information | purified YK23 exhibits high phosphatase activity against fructose 1,6-bisphosphate, significant hydrolytic activity toward fructose 1-phosphate, and detectable activity with glyceraldehyde 3-phosphate, erythrose 4-phosphate, and ribulose 1,5-diphosphate | Saccharomyces cerevisiae | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | the core domain with the alpha/beta/alpha-fold is covered by two small cap domains, structures of presence of two phosphate molecules as inhibitor or FBP (a substrate) bound to the active site, overview | Saccharomyces cerevisiae |
Synonyms | Comment | Organism |
---|---|---|
FBPase | - |
Saccharomyces cerevisiae |
fructose-1,6-bisphosphatase | - |
Saccharomyces cerevisiae |
More | YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily | Saccharomyces cerevisiae |
YK23 | - |
Saccharomyces cerevisiae |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Saccharomyces cerevisiae |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.3 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant S65A | Saccharomyces cerevisiae | |
0.5 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant H178A | Saccharomyces cerevisiae | |
0.7 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant Y24A | Saccharomyces cerevisiae | |
1.6 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant R181A | Saccharomyces cerevisiae | |
1.8 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant R69A | Saccharomyces cerevisiae | |
1.9 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant W131A | Saccharomyces cerevisiae | |
2.8 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant H244A | Saccharomyces cerevisiae | |
2.9 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant S19A | Saccharomyces cerevisiae | |
4.6 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant H268A | Saccharomyces cerevisiae | |
4.9 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, wild-type enzyme | Saccharomyces cerevisiae |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | 6.5 | - |
Saccharomyces cerevisiae |
General Information | Comment | Organism |
---|---|---|
evolution | YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily. YK23 represents the first family of metal-independent FBPases and a second FBPase family in eukaryotes | Saccharomyces cerevisiae |
physiological function | fructose-1,6-bisphosphatase is a key enzyme of gluconeogenesis and photosynthetic CO2 fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways | Saccharomyces cerevisiae |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
300 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant S65A | Saccharomyces cerevisiae | |
340 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant H178A | Saccharomyces cerevisiae | |
1100 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant R181A | Saccharomyces cerevisiae | |
1200 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant H244A | Saccharomyces cerevisiae | |
1200 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant Y24A | Saccharomyces cerevisiae | |
2200 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant W131A | Saccharomyces cerevisiae | |
4100 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant S19A | Saccharomyces cerevisiae | |
5700 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant R69A | Saccharomyces cerevisiae | |
9500 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, mutant H268A | Saccharomyces cerevisiae | |
27000 | - |
D-fructose 1,6-bisphosphate | pH 7.5, 30°C, wild-type enzyme | Saccharomyces cerevisiae |