Literature summary for 2.7.1.40 extracted from

McFarlane, J.S.; Ronnebaum, T.A.; Meneely, K.M.; Chilton, A.; Fenton, A.W.; Lamb, A.L.
Changes in the allosteric site of human liver pyruvate kinase upon activator binding include the breakage of an intersubunit cation-Pi bond (2019), Acta Crystallogr. Sect. F, 75, 461-469 .

Activating Compound

Activating Compound	Comment	Organism	Structure
fructose-1,6-bisphosphate	hLPYK is allosterically activated by fructose-1,6-bisphosphate (Fru-1,6-BP). The allosteric site, as defined by previous structural studies, is located in domain C between the phosphate-binding loop (residues 444-449) and the allosteric loop (residues 527-533). The 6'-phosphate of Fru-1,6-BP contributes to binding by interacting with the phosphate-binding loop (residues 444-449 between beta1 of sheet D and alpha22)	Homo sapiens

Cloned(Commentary)

Cloned (Comment)	Organism
gene pyk, recombinant expression of wild-type and mutant codon-optimized genes as N-terminally His6-tagged enzymes in Escherichia coli strain BL21 Star (DE3)	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant N-terminally His6-tagged mutant enzymes, hanging drop vapour diffusion method, mixing of 0.0015 ml of 2.0-6.0 mg/ml protein in 50 mM MES, pH 6.8, 100 mM KCl, 10% glycerol, and 2 mM DTT, with 0.0015 ml of reservoir solution containing 200 mM ammonium citrate dibasic, and 20% PEG 3350 for mutant D499N, or 325 mM ammonium citrate dibasic, and 16% PEG 3350 for mutant W527H, or 200 mM ammonium citrate, pH 6.0, and 24% PEG 3350 for mutant DELTA529/S531G, or 200 mM ammonium citrate, pH 5.6, and 16% PEG 3350 for mutant S531E, and equilibration against 1 ml of reservoir solution, at 25°C, X-ray diffraction structrue determination and analysis at 2.15-2.42 A resolution, molecular replacement using the A and C domains of chain A of the S12D variant of hLPYK (PDB ID 4ip7) as a rigid-body model, modeling	Homo sapiens

Crystallization (Comment)

Organism

purified recombinant N-terminally His6-tagged mutant enzymes, hanging drop vapour diffusion method, mixing of 0.0015 ml of 2.0-6.0 mg/ml protein in 50 mM MES, pH 6.8, 100 mM KCl, 10% glycerol, and 2 mM DTT, with 0.0015 ml of reservoir solution containing 200 mM ammonium citrate dibasic, and 20% PEG 3350 for mutant D499N, or 325 mM ammonium citrate dibasic, and 16% PEG 3350 for mutant W527H, or 200 mM ammonium citrate, pH 6.0, and 24% PEG 3350 for mutant DELTA529/S531G, or 200 mM ammonium citrate, pH 5.6, and 16% PEG 3350 for mutant S531E, and equilibration against 1 ml of reservoir solution, at 25°C, X-ray diffraction structrue determination and analysis at 2.15-2.42 A resolution, molecular replacement using the A and C domains of chain A of the S12D variant of hLPYK (PDB ID 4ip7) as a rigid-body model, modeling

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
D499N	site-directed mutagenesis, the structure of the D499N mutant does not provide structural evidence for the previously observed allosteric activation of the D499N variant. The increase in PEP affinity observed for the D499N mutant in the absence of Fru-1,6-BP is due to the disruption of allosteric coupling across the C-C interface, crystal structure determination and analysis	Homo sapiens
S531E	site-directed mutagenesis, in the S531E variant glutamate binds in place of the 6'-phosphate of fructose-1,6-bisphosphate in the allosteric site, leading to partial allosteric activation, crystal structure determination and analysis	Homo sapiens
S531G	construction of mutant DELTA529/S531G, the mutant is not activated by Fru-1,6-BP, crystal structure determination and analysis	Homo sapiens
W527H	site-directed mutagenesis, the increase in PEP affinity observed for the W527H mutant in the absence of Fru-1,6-BP is due to the disruption of allosteric coupling across the C-C interface, crystal structure determination and analysis	Homo sapiens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ADP + phosphoenolpyruvate	Homo sapiens	-	ATP + pyruvate	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	P30613	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 Star (DE3) by nickel affinity chromatography and gel filtration	Homo sapiens

Source Tissue

Source Tissue	Comment	Organism	Textmining
liver	-	Homo sapiens	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ADP + phosphoenolpyruvate	-	Homo sapiens	ATP + pyruvate	-	?

Synonyms

Synonyms	Comment	Organism
hLPYK	-	Homo sapiens
liver pyruvate kinase	-	Homo sapiens
PYK	-	Homo sapiens

General Information

General Information	Comment	Organism
malfunction	allosteric site structure analysis of wild-type and mutant enzymes, overview. In the S531E variant glutamate binds in place of the 6'-phosphate of fructose-1,6-bisphosphate in the allosteric site, leading to partial allosteric activation. The structure of the D499N mutant does not provide structural evidence for the previously observed allosteric activation of the D499N variant	Homo sapiens
metabolism	human liver pyruvate kinase (hLPYK) catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate through a phosphoryl transfer from PEP to ADP, generating pyruvate and ATP. In human liver, this penultimate step of glycolysis is allosterically regulated by fructose-1,6-bisphosphate (Fru-1,6-BP), an earlier intermediate of glycolysis	Homo sapiens
additional information	changes in the allosteric site of human liver pyruvate kinase upon activator binding include the breakage of an intersubunit cation-Pi bond. Conformational toggle between the open and closed positions of the allosteric loop, structure analysis of wild-type and mutant enzymes, overview. In the absence of fructose-1,6-bisphosphate the open position is stabilized, in part, by a cation-Pi bond between Trp527 and Arg538' (from an adjacent monomer). In the S531E variant glutamate binds in place of the 6'-phosphate of fructose-1,6-bisphosphate in the allosteric site, leading to partial allosteric activation. The structure of the D499N mutant does not provide structural evidence for the previously observed allosteric activation of the D499N variant	Homo sapiens
physiological function	human liver pyruvate kinase (hLPYK) converts phosphoenolpyruvate to pyruvate in the final step of glycolysis. hLPYK is allosterically activated by fructose-1,6-bisphosphate (Fru-1,6-BP)	Homo sapiens