Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli, expression of all enzyme variants leads to the formation of insoluble inclusion bodies. Refolding of most variants can be followed by measuring increases in enzyme activity | Penicillium amagasakiense |
Protein Variants | Comment | Organism |
---|---|---|
F418A | 12.6fold increase in apparent Km value | Penicillium amagasakiense |
H520A | the enzyme variant is almost completely inactive | Penicillium amagasakiense |
H520V | the enzyme variant is almost completely inactive | Penicillium amagasakiense |
H563A | the enzyme variant is completely inactive | Penicillium amagasakiense |
H563V | the enzyme variant is completely inactive | Penicillium amagasakiense |
additional information | the removal of aromatic or bulky residues at positions 73, 418 or 430 result in decreases in the maximum rates of glucose oxidation to less than 1/90 | Penicillium amagasakiense |
R516K | the mutant enzyme whose side chain forms only one hydrogen bond with the 3-OH group of beta-D-glucose, exhibits an 80fold higher apparent Km (513 mM) but a Vmax only 70% lower than the wild type | Penicillium amagasakiense |
R516Q | the complete elimination of a hydrogen-bond interaction between residue 516 and the 3-OH group of beta-D-glucose through the substitution R516Q effects a 120fold increase in the apparent Km for glucose (to 733 mM) and a decrease in the Vmax to 1/30 | Penicillium amagasakiense |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2 | 4 | beta-D-glucose | 25°C, pH 6.0, mutant enzyme Y73F | Penicillium amagasakiense | |
5.8 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme H520A | Penicillium amagasakiense | |
6.2 | - |
beta-D-glucose | 25°C, pH 6.0, wild-type enzyme | Penicillium amagasakiense | |
6.7 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme H520V | Penicillium amagasakiense | |
13.3 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme F418V | Penicillium amagasakiense | |
27 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme W430A | Penicillium amagasakiense | |
36 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme N518T | Penicillium amagasakiense | |
78 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme F418 | Penicillium amagasakiense | |
513 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme R516K | Penicillium amagasakiense | |
733 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme R516Q | Penicillium amagasakiense |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
60000 | - |
2 * 60000, SDS-PAGE | Penicillium amagasakiense |
120000 | - |
native PAGE | Penicillium amagasakiense |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Penicillium amagasakiense | P81156 | - |
- |
Penicillium amagasakiense ATCC 28686 | P81156 | - |
- |
Purification (Comment) | Organism |
---|---|
all reconstituted variants are purified to homogeneity by mild acidification and ion-exchange chromatography | Penicillium amagasakiense |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
beta-D-glucose + O2 | kinetic studies on the oxidation of beta-D-glucose combined with molecular modelling show the side chain of Arg516, which forms two hydrogen bonds with the 3-OH group of beta-D-glucose, to be absolutely essential for the efficient binding of beta-D-glucose. Of the residues forming the active site of glucose oxidase, Arg516 is the most critical amino acid for the efficient binding of beta-D-glucose by the enzyme, whereas aromatic residues at positions 73, 418 and 430 are important for the correct orientation and maximal velocity of glucose oxidation | Penicillium amagasakiense | D-glucono-1,5-lactone + H2O2 | - |
? | |
beta-D-glucose + O2 | kinetic studies on the oxidation of beta-D-glucose combined with molecular modelling show the side chain of Arg516, which forms two hydrogen bonds with the 3-OH group of beta-D-glucose, to be absolutely essential for the efficient binding of beta-D-glucose. Of the residues forming the active site of glucose oxidase, Arg516 is the most critical amino acid for the efficient binding of beta-D-glucose by the enzyme, whereas aromatic residues at positions 73, 418 and 430 are important for the correct orientation and maximal velocity of glucose oxidation | Penicillium amagasakiense ATCC 28686 | D-glucono-1,5-lactone + H2O2 | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | 2 * 60000, SDS-PAGE | Penicillium amagasakiense |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Penicillium amagasakiense |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.03 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme H520V | Penicillium amagasakiense | |
0.16 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme H520A | Penicillium amagasakiense | |
14 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme F418 | Penicillium amagasakiense | |
23 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme W430A | Penicillium amagasakiense | |
71 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme R516Q | Penicillium amagasakiense | |
318 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme F418V | Penicillium amagasakiense | |
603 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme R516K | Penicillium amagasakiense | |
639 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme Y73F | Penicillium amagasakiense | |
1166 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme N518T | Penicillium amagasakiense | |
2003 | - |
beta-D-glucose | 25°C, pH 6.0, wild-type enzyme | Penicillium amagasakiense |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
assay at | Penicillium amagasakiense |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | dimerization is only possible with the proper incorporation of the cofactor in the FAD-binding pocket | Penicillium amagasakiense |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.004 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme H520V | Penicillium amagasakiense | |
0.03 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme H520A | Penicillium amagasakiense | |
0.1 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme R516Q | Penicillium amagasakiense | |
0.2 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme F418 | Penicillium amagasakiense | |
0.8 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme W430A | Penicillium amagasakiense | |
1.2 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme R516K | Penicillium amagasakiense | |
24 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme F418V | Penicillium amagasakiense | |
26 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme Y73F | Penicillium amagasakiense | |
33 | - |
beta-D-glucose | 25°C, pH 6.0, mutant enzyme N518T | Penicillium amagasakiense | |
323 | - |
beta-D-glucose | 25°C, pH 6.0, wild-type enzyme | Penicillium amagasakiense |