Literature summary for 2.7.1.40 extracted from

Holyoak, T.; Zhang, B.; Deng, J.; Tang, Q.; Prasannan, C.B.; Fenton, A.W.
Energetic coupling between an oxidizable cysteine and the phosphorylatable N-terminus of human liver pyruvate kinase (2013), Biochemistry, 52, 466-476.

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme mutant S12D as S12D-L-PYK-Fru-1,6-bisphosphate-Mn-Na-citrate complex, vapor diffusion hanging drop method, PYK S12D at 4 mg/ml in 10 mM MES, pH 6.8, 5 mM MgCl2, 10 mM KCl, and 2 mM DTT, 52 mM Na-ATP, and 1.3 mM Na-Fru-1,6-BP, mixing with well solution in varied ratios of 2:2, 4:2, 2:4, and 4:4, the well solution contains 50 mM sodium citrate, pH 4.9, 26 mM MnCl2, and 3-5% PEG 6000, X-ray diffraction structure determination and analysis at 1.8-1.95 A resolution, molecular replacement and modeling	Homo sapiens

Crystallization (Comment)

Organism

purified enzyme mutant S12D as S12D-L-PYK-Fru-1,6-bisphosphate-Mn-Na-citrate complex, vapor diffusion hanging drop method, PYK S12D at 4 mg/ml in 10 mM MES, pH 6.8, 5 mM MgCl2, 10 mM KCl, and 2 mM DTT, 52 mM Na-ATP, and 1.3 mM Na-Fru-1,6-BP, mixing with well solution in varied ratios of 2:2, 4:2, 2:4, and 4:4, the well solution contains 50 mM sodium citrate, pH 4.9, 26 mM MnCl2, and 3-5% PEG 6000, X-ray diffraction structure determination and analysis at 1.8-1.95 A resolution, molecular replacement and modeling

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
C436A	site-directed mutagenesis, the mutant shows decreased affinity for phosphoenolpyruvate compared to the wild-type enzyme	Homo sapiens
C436D	site-directed mutagenesis, the mutant shows decreased affinity for phosphoenolpyruvate compared to the wild-type enzyme	Homo sapiens
C436H	site-directed mutagenesis, the mutant shows decreased affinity for phosphoenolpyruvate compared to the wild-type enzyme	Homo sapiens
C436M	site-directed mutagenesis, crystal structure analysis, the mutant of L-PYK is the only residue 436 mutation that strengthens PEP affinity, revealing that the methionine substitution results in the ordering of several N-terminal residues that have not been ordered in previous structures	Homo sapiens
C436N	site-directed mutagenesis, the mutant shows decreased affinity for phosphoenolpyruvate compared to the wild-type enzyme	Homo sapiens
C436S	site-directed mutagenesis, the mutant shows decreased affinity for phosphoenolpyruvate compared to the wild-type enzyme	Homo sapiens
C436T	site-directed mutagenesis, the mutant shows decreased affinity for phosphoenolpyruvate compared to the wild-type enzyme	Homo sapiens
F24A	site-directed mutagenesis, the mutant shows decreased affinity for phosphoenolpyruvate compared to the wild-type enzyme	Homo sapiens
L16A	site-directed mutagenesis, the mutant shows decreased affinity for phosphoenolpyruvate compared to the wild-type enzyme	Homo sapiens
L20A	site-directed mutagenesis, the mutant shows decreased affinity for phosphoenolpyruvate compared to the wild-type enzyme	Homo sapiens
Q18A	site-directed mutagenesis, the mutant shows strengthened phosphoenolpyruvate affinity compared to the wild-type enzyme	Homo sapiens
S12A	site-directed mutagenesis, the mutant shows strengthened phosphoenolpyruvate affinity compared to the wild-type enzyme	Homo sapiens
S12D	site-directed mutagenesis, the S12D mutation mimics the effect of phosphorylation on L-PYK function, crystal structure analysis, overview	Homo sapiens
T22A	site-directed mutagenesis, the mutant shows strengthened phosphoenolpyruvate affinity compared to the wild-type enzyme	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism	Structure
L-alanine	-	Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	phosphoenolpyruvate affinity is sensitive to the nature of the side chain at position 436	Homo sapiens
0.112	-	phosphoenolpyruvate	mutant C436M, pH not specified in the publication, 30°C	Homo sapiens
0.24	-	phosphoenolpyruvate	wild-type enzyme, pH not specified in the publication, 30°C	Homo sapiens
0.345	-	phosphoenolpyruvate	mutant C436H, pH not specified in the publication, 30°C	Homo sapiens
0.372	-	phosphoenolpyruvate	mutant C436A, pH not specified in the publication, 30°C	Homo sapiens
0.552	-	phosphoenolpyruvate	mutant S12D, pH not specified in the publication, 30°C	Homo sapiens
0.703	-	phosphoenolpyruvate	mutant C436N, pH not specified in the publication, 30°C	Homo sapiens
0.768	-	phosphoenolpyruvate	mutant C436T, pH not specified in the publication, 30°C	Homo sapiens
0.802	-	phosphoenolpyruvate	mutant C436S, pH not specified in the publication, 30°C	Homo sapiens
0.804	-	phosphoenolpyruvate	mutant C436D, pH not specified in the publication, 30°C	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.
ATP + pyruvate	Homo sapiens	-	ADP + phosphoenolpyruvate	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	P30613	isozyme L	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
phosphoprotein	the phosphorylated N-terminus does not interact with the main body of the protein or does so with a very low binding energy	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.
ATP + pyruvate	-	Homo sapiens	ADP + phosphoenolpyruvate	-	?

Synonyms

Synonyms	Comment	Organism
L-PYK	-	Homo sapiens

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Homo sapiens

General Information

General Information	Comment	Organism
malfunction	human liver pyruvate kinase shows reduced affinity for phosphoenolpyruvate several days after cell lysis because Cys436 is oxidized, an effect of aging. The side chain of residue 436 is energetically coupled to phosphoenolpyruvate binding, overview	Homo sapiens
additional information	energetic coupling between an oxidizable cysteine and the phosphorylatable N-terminus of human liver pyruvate kinase determines substrate affinity and activity, overview. Oxidation of Cys436 and phosphorylation of the N-terminus at Ser12 may function through a similar mechanism, namely the interruption of an activating interaction between the nonphosphorylated N-terminus with the nonoxidized main body of the protein. Modeling of C436M-L-PYK-citrate-Mn-ATP-Fru-1,6-bisphosphate complex using crystal structure of S12D mutant in a S12D-L-PYK-Fru-1,6-bisphosphate-Mn-Na-citrate complex, overview	Homo sapiens