Literature summary for 2.7.1.146 extracted from

Currie, M.A.; Merino, F.; Skarina, T.; Wong, A.H.; Singer, A.; Brown, G.; Savchenko, A.; Caniuguir, A.; Guixe, V.; Yakunin, A.F.; Jia, Z.
ADP-dependent 6-phosphofructokinase from Pyrococcus horikoshii OT3: structure determination and biochemical characterization of PH1645 (2009), J. Biol. Chem., 284, 22664-22671.

Search for more literature for 2.7.1.146

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli BL21(DE3) and DL41(DE3) cells	Pyrococcus horikoshii
recombinant wild type and mutant native and selenomethionine-labeled proteins are expressed in BL21(DE3) and DL41(DE3) Escherichia coli cells	Pyrococcus horikoshii

Crystallization (Commentary)

Crystallization (Comment)	Organism
hanging drop vapor diffusion method, the apo-enzyme is crystallized using 22% PEG 4000, 0.1 M Tris-HCl, pH 8.5, and 0.2 M LiSO4, at 21°C. Crystals of the PFK complex with AMP are obtained by crystallization of the PFK D17A protein in the presence of 20% PEG 3350, 0.2 M lithium citrate, 10 mM D-fructose 6-phosphate, and 5 mM ADP	Pyrococcus horikoshii
through hanging drop vapor diffusion at 21°C by mixing protein solution with solution consisting of 22% PEG 4000, 0.1 M Tris-HCl, pH 8.5, and 0.2 M Li2SO4, crystals of the ADP-dependent 6-phosphofructokinase complex with AMP are obtained by crystallization of the mutant protein D17A protein in the presence of 20% PEG 3350, 0.2 M lithium citrate, 10 mM fructose 6-phosphate, and 5 mM ADPcrystal structure of ADP-dependent 6-phosphofructokinase in both apo- and AMP-bound forms determined to 2.0 A and 1.9 A resolution	Pyrococcus horikoshii

Crystallization (Comment)

Organism

hanging drop vapor diffusion method, the apo-enzyme is crystallized using 22% PEG 4000, 0.1 M Tris-HCl, pH 8.5, and 0.2 M LiSO4, at 21°C. Crystals of the PFK complex with AMP are obtained by crystallization of the PFK D17A protein in the presence of 20% PEG 3350, 0.2 M lithium citrate, 10 mM D-fructose 6-phosphate, and 5 mM ADP

Pyrococcus horikoshii

through hanging drop vapor diffusion at 21°C by mixing protein solution with solution consisting of 22% PEG 4000, 0.1 M Tris-HCl, pH 8.5, and 0.2 M Li2SO4, crystals of the ADP-dependent 6-phosphofructokinase complex with AMP are obtained by crystallization of the mutant protein D17A protein in the presence of 20% PEG 3350, 0.2 M lithium citrate, 10 mM fructose 6-phosphate, and 5 mM ADPcrystal structure of ADP-dependent 6-phosphofructokinase in both apo- and AMP-bound forms determined to 2.0 A and 1.9 A resolution

Pyrococcus horikoshii

Protein Variants

Protein Variants	Comment	Organism
A71E	production by site-directed mutagenesis	Pyrococcus horikoshii
A71E	the mutant shows slightly decreased kcat compared to the wild type enzyme	Pyrococcus horikoshii
D17A	production by site-directed mutagenesis	Pyrococcus horikoshii
D17A	the mutant shows severely decreased kcat compared to the wild type enzyme	Pyrococcus horikoshii
D433A	production by site-directed mutagenesis, loss of PFK activity	Pyrococcus horikoshii
D433A	the mutant shows no acitivity	Pyrococcus horikoshii
K158A	production by site-directed mutagenesis	Pyrococcus horikoshii
K158A	the mutant shows slightly decreased kcat compared to the wild type enzyme	Pyrococcus horikoshii
N15A	production by site-directed mutagenesis	Pyrococcus horikoshii
N15A	the mutant shows increased kcat compared to the wild type enzyme	Pyrococcus horikoshii
N160A	production by site-directed mutagenesis	Pyrococcus horikoshii
N160A	the mutant shows strongly increased kcat compared to the wild type enzyme	Pyrococcus horikoshii
Q224A	production by site-directed mutagenesis	Pyrococcus horikoshii
Q224A	the mutant shows increased kcat compared to the wild type enzyme	Pyrococcus horikoshii
Q97A	production by site-directed mutagenesis	Pyrococcus horikoshii
Q97A	the mutant shows decreased kcat compared to the wild type enzyme	Pyrococcus horikoshii
R185A	production by site-directed mutagenesis	Pyrococcus horikoshii
R185A	the mutant shows no acitivity	Pyrococcus horikoshii
R191A	production by site-directed mutagenesis	Pyrococcus horikoshii
R191A	the mutant shows decreased kcat compared to the wild type enzyme	Pyrococcus horikoshii
R191E	production by site-directed mutagenesis	Pyrococcus horikoshii
R191E	the mutant shows almost wild type enzyme activity	Pyrococcus horikoshii
S183A	the mutant shows strongly increased kcat compared to the wild type enzyme	Pyrococcus horikoshii
S183E	production by site-directed mutagenesis, the S183E mutant displays significantly less activity when the phosphoryl donor is replaced with IDP, UDP, GDP, or CDP compared with the wild type	Pyrococcus horikoshii
S183E	the mutant displays significantly less activity when the phosphoryl donor ADP is replaced with IDP, UDP, GDP, or CDP, compared with the wild type	Pyrococcus horikoshii
S189A	production by site-directed mutagenesis	Pyrococcus horikoshii
S189A	the mutant shows decreased kcat compared to the wild type enzyme	Pyrococcus horikoshii
S263A	production by site-directed mutagenesis	Pyrococcus horikoshii
S263A	the mutant shows increased kcat compared to the wild type enzyme	Pyrococcus horikoshii

Inhibitors

Inhibitors	Comment	Organism
Ca2+	about 50% activity at 2.5 mM	Pyrococcus horikoshii
Mg2+	about 60% activity at 2.5 mM	Pyrococcus horikoshii
Mn2+	about 70% activity at 2.5 mM	Pyrococcus horikoshii

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.00395	-	D-fructose 6-phosphate	A71E mutant, at 50°C	Pyrococcus horikoshii
0.00395	-	D-fructose 6-phosphate	mutant enzyme A71E, at 50°C	Pyrococcus horikoshii
0.00708	-	D-fructose 6-phosphate	Q97A mutant, at 50°C	Pyrococcus horikoshii
0.00708	-	D-fructose 6-phosphate	mutant enzyme Q97A, at 50°C	Pyrococcus horikoshii
0.00747	-	ADP	Q97A mutant, at 50°C	Pyrococcus horikoshii
0.00747	-	ADP	mutant enzyme Q97A, at 50°C	Pyrococcus horikoshii
0.00902	-	D-fructose 6-phosphate	S189A mutant, at 50°C	Pyrococcus horikoshii
0.00902	-	D-fructose 6-phosphate	mutant enzyme S189A, at 50°C	Pyrococcus horikoshii
0.0127	-	D-fructose 6-phosphate	D433A mutant, at 50°C	Pyrococcus horikoshii
0.0127	-	D-fructose 6-phosphate	mutant enzyme D433A, at 50°C	Pyrococcus horikoshii
0.0132	-	ADP	wild type enzyme, at 50°C	Pyrococcus horikoshii
0.0136	-	ADP	A71E mutant, at 50°C	Pyrococcus horikoshii
0.0136	-	ADP	mutant enzyme A71E, at 50°C	Pyrococcus horikoshii
0.0139	-	ADP	D433A mutant, at 50°C	Pyrococcus horikoshii
0.0139	-	ADP	mutant enzyme D433A, at 50°C	Pyrococcus horikoshii
0.0145	-	ADP	R191A mutant, at 50°C	Pyrococcus horikoshii
0.0145	-	ADP	mutant enzyme R191A, at 50°C	Pyrococcus horikoshii
0.0152	-	D-fructose 6-phosphate	wild type enzyme, at 50°C	Pyrococcus horikoshii
0.016	-	ADP	S189A mutant, at 50°C	Pyrococcus horikoshii
0.016	-	ADP	mutant enzyme S189A, at 50°C	Pyrococcus horikoshii
0.0208	-	ADP	S263A mutant, at 50°C	Pyrococcus horikoshii
0.0208	-	ADP	mutant enzyme S263A, at 50°C	Pyrococcus horikoshii
0.0238	-	ADP	N15A mutant, at 50°C	Pyrococcus horikoshii
0.0238	-	ADP	mutant enzyme N15A, at 50°C	Pyrococcus horikoshii
0.0295	-	ADP	Q224A mutant, at 50°C	Pyrococcus horikoshii
0.0295	-	ADP	mutant enzyme Q224A, at 50°C	Pyrococcus horikoshii
0.0296	-	ADP	S183E mutant, at 50°C	Pyrococcus horikoshii
0.0296	-	ADP	mutant enzyme S183E, at 50°C	Pyrococcus horikoshii
0.0379	-	ADP	K158A mutant, at 50°C	Pyrococcus horikoshii
0.0379	-	ADP	mutant enzyme K158A, at 50°C	Pyrococcus horikoshii
0.0386	-	D-fructose 6-phosphate	Q224A mutant, at 50°C	Pyrococcus horikoshii
0.0386	-	D-fructose 6-phosphate	mutant enzyme Q224A, at 50°C	Pyrococcus horikoshii
0.0395	-	D-fructose 6-phosphate	S263A mutant, at 50°C	Pyrococcus horikoshii
0.0395	-	D-fructose 6-phosphate	mutant enzyme S263A, at 50°C	Pyrococcus horikoshii
0.0605	-	ADP	R191E mutant, at 50°C	Pyrococcus horikoshii
0.0605	-	ADP	mutant enzyme R191E, at 50°C	Pyrococcus horikoshii
0.0724	-	ADP	N160A mutant, at 50°C	Pyrococcus horikoshii
0.0724	-	ADP	mutant enzyme N160A, at 50°C	Pyrococcus horikoshii
0.0865	-	D-fructose 6-phosphate	S183E mutant, at 50°C	Pyrococcus horikoshii
0.0865	-	D-fructose 6-phosphate	mutant enzyme S183E, at 50°C	Pyrococcus horikoshii
0.103	-	D-fructose 6-phosphate	N15A mutant, at 50°C	Pyrococcus horikoshii
0.103	-	D-fructose 6-phosphate	mutant enzyme N15A, at 50°C	Pyrococcus horikoshii
0.2544	-	D-fructose 6-phosphate	R191A mutant, at 50°C	Pyrococcus horikoshii
0.2544	-	D-fructose 6-phosphate	mutant enzyme R191A, at 50°C	Pyrococcus horikoshii
0.318	-	D-fructose 6-phosphate	R185A mutant, at 50°C	Pyrococcus horikoshii
0.318	-	D-fructose 6-phosphate	mutant enzyme R185A, at 50°C	Pyrococcus horikoshii
0.415	-	D-fructose 6-phosphate	N160A mutant, at 50°C	Pyrococcus horikoshii
0.415	-	D-fructose 6-phosphate	mutant enzyme N160A, at 50°C	Pyrococcus horikoshii
0.473	-	ADP	R185A mutant, at 50°C	Pyrococcus horikoshii
0.473	-	ADP	mutant enzyme R185A, at 50°C	Pyrococcus horikoshii
4.87	-	D-fructose 6-phosphate	R191E mutant, at 50°C	Pyrococcus horikoshii
4.87	-	D-fructose 6-phosphate	mutant enzyme R191E, at 50°C	Pyrococcus horikoshii
6.5	-	D-fructose 6-phosphate	K158A mutant, at 50°C	Pyrococcus horikoshii
22.6	-	D-fructose 6-phosphate	D17A mutant, at 50°C	Pyrococcus horikoshii
22.6	-	D-fructose 6-phosphate	mutant enzyme D17A, at 50°C	Pyrococcus horikoshii
65	-	D-fructose 6-phosphate	mutant enzyme K158A, at 50°C	Pyrococcus horikoshii

Metals/Ions

Metals/Ions	Comment	Organism
CaCl2	2 mM ADP and 2 mM D-fructose 6-phosphate, CaCl2 show the lowest activity with 75% of the activity measured in the presence of MgCl2, activity does not change with the cation concentration in the range of 2.5 to 7 mM	Pyrococcus horikoshii
MgSO4	2 mM ADP and 2 mM D-fructose 6-phosphate, magnesium is tested with 2 counter ions to discard any effect of the anion	Pyrococcus horikoshii
MnCl2	2 mM ADP and 2 mM D-fructose 6-phosphate, increase in enzyme activity with the increase in MnCl2 concentration from 2.5 to 7 mM is observed	Pyrococcus horikoshii
additional information	no significant activity is detected in the presence of Zn2+	Pyrococcus horikoshii
Ni2+	highest activity in the presence of 2.5 mM NiSO4	Pyrococcus horikoshii
NiSO4	2 mM ADP and 2 mM D-fructose 6-phosphate, highest PhPFK activity is obtained with NiSO4 (97 units/mg), decrease in enzyme activity with the increase in MnCl2 concentration from 2.5 to 7 mM is observed	Pyrococcus horikoshii
ZnCl2	2 mM ADP and 2 mM D-fructose 6-phosphate, no significant activity	Pyrococcus horikoshii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ADP + D-fructose 6-phosphate	Pyrococcus horikoshii	-	AMP + D-fructose 1,6-bisphosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus horikoshii	-	-	-
Pyrococcus horikoshii	O59355	strain OT3	-
Pyrococcus horikoshii OT-3	O59355	strain OT3	-

Purification (Commentary)

Purification (Comment)	Organism
by using nickel-nitrilotriacetic acid affinity chromatography and gel filtration	Pyrococcus horikoshii
Ni-NTA column chromatography	Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ADP + D-fructose 6-phosphate	-	Pyrococcus horikoshii	AMP + D-fructose 1,6-bisphosphate	-	?
D-fructose 6-phosphate + ADP	100% activity	Pyrococcus horikoshii	D-fructose 1,6-bisphosphate + AMP	-	?
D-fructose 6-phosphate + ADP	100% activity	Pyrococcus horikoshii OT-3	D-fructose 1,6-bisphosphate + AMP	-	?
D-fructose 6-phosphate + CDP	about 35% activity compared to ADP	Pyrococcus horikoshii	D-fructose 1,6-bisphosphate + CMP	-	?
D-fructose 6-phosphate + CDP	about 35% activity compared to ADP	Pyrococcus horikoshii OT-3	D-fructose 1,6-bisphosphate + CMP	-	?
D-fructose 6-phosphate + GDP	about 50% activity compared to ADP	Pyrococcus horikoshii	D-fructose 1,6-bisphosphate + GMP	-	?
D-fructose 6-phosphate + GDP	about 50% activity compared to ADP	Pyrococcus horikoshii OT-3	D-fructose 1,6-bisphosphate + GMP	-	?
D-fructose 6-phosphate + IDP	about 50% activity compared to ADP	Pyrococcus horikoshii	D-fructose 1,6-bisphosphate + IMP	-	?
D-fructose 6-phosphate + IDP	about 50% activity compared to ADP	Pyrococcus horikoshii OT-3	D-fructose 1,6-bisphosphate + IMP	-	?
D-fructose 6-phosphate + UDP	about 105% activity compared to ADP	Pyrococcus horikoshii	D-fructose 1,6-bisphosphate + UMP	-	?
D-fructose 6-phosphate + UDP	about 105% activity compared to ADP	Pyrococcus horikoshii OT-3	D-fructose 1,6-bisphosphate + UMP	-	?

Synonyms

Synonyms	Comment	Organism
ADP-dependent 6-phosphofructokinase	-	Pyrococcus horikoshii
ADP-Pfk	-	Pyrococcus horikoshii
PhPFK	-	Pyrococcus horikoshii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
50	-	assay at	Pyrococcus horikoshii

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0048	-	D-fructose 6-phosphate	D433A mutant	Pyrococcus horikoshii
0.0048	-	D-fructose 6-phosphate	mutant enzyme D433A, at 50°C	Pyrococcus horikoshii
0.31	-	D-fructose 6-phosphate	R185A mutant	Pyrococcus horikoshii
0.351	-	D-fructose 6-phosphate	mutant enzyme R185A, at 50°C	Pyrococcus horikoshii
1.98	-	D-fructose 6-phosphate	D17A mutant	Pyrococcus horikoshii
1.98	-	D-fructose 6-phosphate	mutant enzyme D17A, at 50°C	Pyrococcus horikoshii
13	-	D-fructose 6-phosphate	Q97A mutant	Pyrococcus horikoshii
13	-	D-fructose 6-phosphate	mutant enzyme Q97A, at 50°C	Pyrococcus horikoshii
25.5	-	D-fructose 6-phosphate	S189A mutant	Pyrococcus horikoshii
25.5	-	D-fructose 6-phosphate	mutant enzyme S189A, at 50°C	Pyrococcus horikoshii
27.4	-	D-fructose 6-phosphate	R191A mutant	Pyrococcus horikoshii
27.4	-	D-fructose 6-phosphate	mutant enzyme R191A, at 50°C	Pyrococcus horikoshii
39.3	-	D-fructose 6-phosphate	A71E mutant	Pyrococcus horikoshii
39.3	-	D-fructose 6-phosphate	mutant enzyme A71E, at 50°C	Pyrococcus horikoshii
41	-	D-fructose 6-phosphate	K158A mutant	Pyrococcus horikoshii
41	-	D-fructose 6-phosphate	mutant enzyme K158A, at 50°C	Pyrococcus horikoshii
42.5	-	D-fructose 6-phosphate	R191E mutant	Pyrococcus horikoshii
42.5	-	D-fructose 6-phosphate	mutant enzyme R191E, at 50°C	Pyrococcus horikoshii
45.5	-	D-fructose 6-phosphate	wild type enzyme, at 50°C	Pyrococcus horikoshii
64.1	-	D-fructose 6-phosphate	S263A mutant	Pyrococcus horikoshii
64.1	-	D-fructose 6-phosphate	mutant enzyme S263A, at 50°C	Pyrococcus horikoshii
65.3	-	D-fructose 6-phosphate	Q224A mutant	Pyrococcus horikoshii
65.3	-	D-fructose 6-phosphate	mutant enzyme Q224A, at 50°C	Pyrococcus horikoshii
91.1	-	D-fructose 6-phosphate	N15A mutant	Pyrococcus horikoshii
91.1	-	D-fructose 6-phosphate	mutant enzyme N15A, at 50°C	Pyrococcus horikoshii
91.7	-	D-fructose 6-phosphate	S183E mutant	Pyrococcus horikoshii
91.7	-	D-fructose 6-phosphate	mutant enzyme S183E, at 50°C	Pyrococcus horikoshii
151	-	D-fructose 6-phosphate	N160A mutant	Pyrococcus horikoshii
151	-	D-fructose 6-phosphate	mutant enzyme N160A, at 50°C	Pyrococcus horikoshii

Cofactor

Cofactor	Comment	Organism
ADP	-	Pyrococcus horikoshii
CDP	-	Pyrococcus horikoshii
GDP	-	Pyrococcus horikoshii
IDP	-	Pyrococcus horikoshii
UDP	-	Pyrococcus horikoshii

General Information

General Information	Comment	Organism
metabolism	Embden-Meyerhof glycolytic pathway, early step	Pyrococcus horikoshii

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
additional information	-	ADP	the calculated value for UDP is 40times higher than that of ADP, indicating a strong preference for ADP over UDP as the phosphoryl group donor	Pyrococcus horikoshii
0.0876	-	D-fructose 6-phosphate	mutant enzyme D17A, at 50°C	Pyrococcus horikoshii
0.345	-	ADP	mutant enzyme D433A, at 50°C	Pyrococcus horikoshii
0.378	-	D-fructose 6-phosphate	mutant enzyme D433A, at 50°C	Pyrococcus horikoshii
0.742	-	ADP	mutant enzyme R185A, at 50°C	Pyrococcus horikoshii
1.1	-	D-fructose 6-phosphate	mutant enzyme R185A, at 50°C	Pyrococcus horikoshii
6.31	-	D-fructose 6-phosphate	mutant enzyme K158A, at 50°C	Pyrococcus horikoshii
8.73	-	D-fructose 6-phosphate	mutant enzyme R191E, at 50°C	Pyrococcus horikoshii
107	-	D-fructose 6-phosphate	mutant enzyme R191A, at 50°C	Pyrococcus horikoshii
364	-	D-fructose 6-phosphate	mutant enzyme N160A, at 50°C	Pyrococcus horikoshii
702	-	ADP	mutant enzyme R191E, at 50°C	Pyrococcus horikoshii
884	-	D-fructose 6-phosphate	mutant enzyme N15A, at 50°C	Pyrococcus horikoshii
1060	-	D-fructose 6-phosphate	mutant enzyme S183E, at 50°C	Pyrococcus horikoshii
1080	-	ADP	mutant enzyme K158A, at 50°C	Pyrococcus horikoshii
1600	-	ADP	mutant enzyme S189A, at 50°C	Pyrococcus horikoshii
1620	-	D-fructose 6-phosphate	mutant enzyme S263A, at 50°C	Pyrococcus horikoshii
1690	-	D-fructose 6-phosphate	mutant enzyme Q224A, at 50°C	Pyrococcus horikoshii
1720	-	D-fructose 6-phosphate	mutant enzyme A71E, at 50°C	Pyrococcus horikoshii
1740	-	ADP	mutant enzyme Q97A, at 50°C	Pyrococcus horikoshii
1840	-	D-fructose 6-phosphate	mutant enzyme Q97A, at 50°C	Pyrococcus horikoshii
1890	-	ADP	mutant enzyme R191A, at 50°C	Pyrococcus horikoshii
2090	-	ADP	mutant enzyme N160A, at 50°C	Pyrococcus horikoshii
2210	-	ADP	mutant enzyme Q224A, at 50°C	Pyrococcus horikoshii
2820	-	ADP	mutant enzyme A71E, at 50°C	Pyrococcus horikoshii
2830	-	D-fructose 6-phosphate	mutant enzyme S189A, at 50°C	Pyrococcus horikoshii
2990	-	D-fructose 6-phosphate	wild type enzyme, at 50°C	Pyrococcus horikoshii
3080	-	ADP	mutant enzyme S263A, at 50°C	Pyrococcus horikoshii
3100	-	ADP	mutant enzyme S183E, at 50°C	Pyrococcus horikoshii
3450	-	ADP	wild type enzyme, at 50°C	Pyrococcus horikoshii
3830	-	ADP	mutant enzyme N15A, at 50°C	Pyrococcus horikoshii