Literature summary for 3.2.1.22 extracted from

Merceron, R.; Foucault, M.; Haser, R.; Mattes, R.; Watzlawick, H.; Gouet, P.
The molecular mechanism of thermostable beta-galactosidases AgaA and AgaB explained by X-ray crystallography and mutational studies (2012), J. Biol. Chem., 287, 39642-39652.

Search for more literature for 3.2.1.22

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli RM448 cells	Geobacillus stearothermophilus

Crystallization (Commentary)

Crystallization (Comment)	Organism
vapor diffusion method, using 80 mM sodium acetate, 20% (w/v) polyethylene glycol 4000, and 6.4% (v/v) isopropanol	Geobacillus stearothermophilus

Protein Variants

Protein Variants	Comment	Organism
W336A	the mutant shows about 28fold reduced catalytic efficiency compared to the wild type	Geobacillus stearothermophilus
W336D	the mutant shows severely reduced catalytic efficiency compared to the wild type	Geobacillus stearothermophilus
W336F	the mutant shows about 3fold increased catalytic efficiency compared to the wild type	Geobacillus stearothermophilus
W336N	the mutant shows about 10fold reduced catalytic efficiency compared to the wild type	Geobacillus stearothermophilus
W336S	the mutant shows about 100fold reduced catalytic efficiency compared to the wild type	Geobacillus stearothermophilus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.21	-	4-nitrophenyl alpha-D-galactopyranoside	wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
0.24	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
0.44	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
0.83	-	4-nitrophenyl alpha-D-galactopyranoside	wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
0.84	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
1.33	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336D of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
3.8	-	raffinose	wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
8.7	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
36.6	-	raffinose	mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
200	-	raffinose	wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
205	-	raffinose	mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
250	-	raffinose	mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
252	-	raffinose	mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus

Organism

Organism	UniProt	Comment	Textmining
Geobacillus stearothermophilus	Q9ALJ4	-	-

Purification (Commentary)

Purification (Comment)	Organism
DMAE column chromatography, Mono Q column chromatography and Superdex 200 gel filtration	Geobacillus stearothermophilus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
4-nitrophenyl alpha-D-galactopyranoside + H2O	-	Geobacillus stearothermophilus	4-nitrophenol + alpha-D-galactopyranose	-	?
raffinose + H2O	-	Geobacillus stearothermophilus	?	-	?

Synonyms

Synonyms	Comment	Organism
AgaA	isoform	Geobacillus stearothermophilus
AgaB	isoform	Geobacillus stearothermophilus
Alpha-D-galactoside galactohydrolase	-	Geobacillus stearothermophilus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.97	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336D of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
5.2	-	raffinose	mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
7.9	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
10.3	-	raffinose	mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
38.2	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
48	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
48.1	-	raffinose	mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
105.6	-	4-nitrophenyl alpha-D-galactopyranoside	wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
180	-	raffinose	wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
375	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
832	-	raffinose	mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
1205	-	raffinose	wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
2382	-	4-nitrophenyl alpha-D-galactopyranoside	wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
0.05	-	raffinose	mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
0.14	-	raffinose	mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
0.19	-	raffinose	mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
0.7	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336D of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
3.3	-	raffinose	mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
5.5	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
6	-	raffinose	wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
17.9	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
45.5	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
64.3	-	raffinose	wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
502	-	4-nitrophenyl alpha-D-galactopyranoside	wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
1562	-	4-nitrophenyl alpha-D-galactopyranoside	mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus
2870	-	4-nitrophenyl alpha-D-galactopyranoside	wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C	Geobacillus stearothermophilus

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Release 2023.1 (January 2023)