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Literature summary for 1.1.3.4 extracted from

  • Witt, S.; Wohlfahrt, G.; Schomburg, D.; Hecht, H.-J.; Kalisz, H.M.
    Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of beta-D-glucose (2000), Biochem. J., 347, 553-559.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

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 [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 [Da] Molecular Weight Maximum [Da] Comment Organism
60000
-
2 * 60000, SDS-PAGE Penicillium amagasakiense
120000
-
native PAGE Penicillium amagasakiense

Organism

Organism UniProt Comment Textmining
Penicillium amagasakiense P81156
-
-
Penicillium amagasakiense ATCC 28686 P81156
-
-

Purification (Commentary)

Purification (Comment) Organism
all reconstituted variants are purified to homogeneity by mild acidification and ion-exchange chromatography Penicillium amagasakiense

Substrates and Products (Substrate)

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

Subunits Comment Organism
dimer 2 * 60000, SDS-PAGE Penicillium amagasakiense

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Penicillium amagasakiense

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
assay at Penicillium amagasakiense

Cofactor

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 [mM/s]

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