Literature summary for 2.7.1.40 extracted from

Morgan, H.P.; Walsh, M.J.; Blackburn, E.A.; Wear, M.A.; Boxer, M.B.; Shen, M.; Veith, H.; McNae, I.W.; Nowicki, M.W.; Michels, P.A.; Auld, D.S.; Fothergill-Gilmore, L.A.; Walkinshaw, M.D.
A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase (2012), Biochem. J., 448, 67-72.

Cloned(Commentary)

Cloned (Comment)	Organism
expression of wild-type and mutant enzymes in Escherichia coli strain Rosetta 2 (DE3)pLysS	Leishmania mexicana

Crystallization (Commentary)

Crystallization (Comment)	Organism
enzyme complexed with inhibitor 4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid, mxing of 0.0015 ml of 10 mg/ml protein in 20 mM TEA, pH 7.2, 1 mM 1,3,6,8-pyrenetetrasulfonic acid, and 9 mM inhibitor, with 0.0015 ml of well solution containing 12-16% PEG 8000, 20 mM TEA buffer, pH 7.2, 50 mM magnesium chloride, 100 mM potassium chloride, and 10% glycerol, 1-2 days, and equilibration for 14 h over a well solution composed of 14-18% PEG 8,000, 20 mM TEA buffer, pH 7.2, 50 mM magnesium chloride, 100 mM potassium chloride, and 25% glycerol, X-ray diffraction structure determination and analysis at 2.65 A resolution	Leishmania mexicana

Crystallization (Comment)

Organism

enzyme complexed with inhibitor 4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid, mxing of 0.0015 ml of 10 mg/ml protein in 20 mM TEA, pH 7.2, 1 mM 1,3,6,8-pyrenetetrasulfonic acid, and 9 mM inhibitor, with 0.0015 ml of well solution containing 12-16% PEG 8000, 20 mM TEA buffer, pH 7.2, 50 mM magnesium chloride, 100 mM potassium chloride, and 10% glycerol, 1-2 days, and equilibration for 14 h over a well solution composed of 14-18% PEG 8,000, 20 mM TEA buffer, pH 7.2, 50 mM magnesium chloride, 100 mM potassium chloride, and 25% glycerol, X-ray diffraction structure determination and analysis at 2.65 A resolution

Leishmania mexicana

Protein Variants

Protein Variants	Comment	Organism
K335R	site-directed mutagenesis, structure compared to the wild-type, overview	Leishmania mexicana

Inhibitors

Inhibitors	Comment	Organism
3-(2,5-dimethylphenoxy)-1,2-benzothiazole 1,1-dioxide	a saccharin derivative, potent inhibitor, but a labile compound	Leishmania mexicana
3-[(2,5-dimethylphenyl)sulfanyl]-1,2-benzothiazole 1,1-dioxide	a stable sulfur derivative of 3-(2,5-dimethylphenoxy)-1,2-benzothiazole 1,1-dioxide	Leishmania mexicana
4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid	irreversible inhibitor, a saccharin derivative, reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP, covalent inhibitor mechanism, overview. Inhibition of LmPYK by the compound is time-dependent	Leishmania mexicana
additional information	the nitrogen analogue N-(2,5-dimethylphenyl)-1,2-benzothiazol-3-amine 1,1-dioxide of 3-(2,5-dimethylphenoxy)-1,2-benzothiazole 1,1-dioxide is not inhibitory	Leishmania mexicana

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Leishmania mexicana

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Leishmania mexicana	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain Rosetta 2 (DE3)pLysS	Leishmania mexicana

Substrates and Products (Substrate)

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

Synonyms

Synonyms	Comment	Organism
PYK	-	Leishmania mexicana

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Leishmania mexicana

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.2	-	assay at	Leishmania mexicana

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Leishmania mexicana

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor	Structure
0.005	-	pH 7.2, 25°C	Leishmania mexicana	3-[(2,5-dimethylphenyl)sulfanyl]-1,2-benzothiazole 1,1-dioxide
0.01	-	pH 7.2, 25°C	Leishmania mexicana	3-(2,5-dimethylphenoxy)-1,2-benzothiazole 1,1-dioxide

General Information

General Information	Comment	Organism
physiological function	PYK plays a central role in a number of proliferative and infectious diseases	Leishmania mexicana