Biomaterial Database

Synthesis and biochemical characterization of the new sulfhydryl-reactive ATP analogue 8-thiocyano-ATP. Its interaction with Na,K-ATPase and kinases. 1992

G Scheiner-Bobis, and W Mertens, and M Willeke, and W Schoner

Institut für Biochemie, Justus-Liebig-Universität Giessen, Germany.

The synthesis of 8-thiocyano-ATP (CNS8-ATP) is described. At 37 degrees C the ATP analogue inactivates Na,K-ATPase, hexokinase, and pyruvate kinase. In all three cases, inactivation can be prevented by the addition of ATP, thus indicating that CNS8-ATP is recognized within the ATP binding site of the above enzymes. Incubation of the inactivated enzymes with dithiothreitol restores the catalytic activities. Therefore, it is likely that in these enzymes a mixed disulfide (E-S-S8-ATP) is formed between a sulfhydryl in the ATP binding site (E-SH) and the ATP analogue: [formula: see text] From the pseudo-first-order inactivation kinetics, a KD = 2.7 microM with k2 = 0.142 min-1 is calculated for the hexokinase and a KD = 40 microM with k2 = 0.347 min-1 is calculated for the pyruvate kinase interactions with the ATP analogue. At 4 degrees C, Na,K-ATPase recognizes CNS8-ATP with a KD = 8.3 microM. At 37 degrees C, the enzyme becomes inactivated by the ATP analogue in a biphasic manner. Inactivation results in the incorporation of [alpha-32P]8-CNS8-ATP into the catalytic alpha-subunit of the enzyme. Limited tryptic digestion in the presence of 150 mM KCl results in the formation of a radioactive peptide of Mr = 56,000, known to bear the purine binding domain of Na,K-ATPase. The results described in this article verify CNS8-ATP as a sulfhydryl-reactive ATP analogue and characterize this new ATP analogue as a useful tool for structure/function studies on ATP-recognizing enzymes.

UI	MeSH Term	Description	Entries
D011770	Pyruvate Kinase	ATP:pyruvate 2-O-phosphotransferase. A phosphotransferase that catalyzes reversibly the phosphorylation of pyruvate to phosphoenolpyruvate in the presence of ATP. It has four isozymes (L, R, M1, and M2). Deficiency of the enzyme results in hemolytic anemia. EC 2.7.1.40.	L-Type Pyruvate Kinase,M-Type Pyruvate Kinase,M1-Type Pyruvate Kinase,M2-Type Pyruvate Kinase,Pyruvate Kinase L,R-Type Pyruvate Kinase,L Type Pyruvate Kinase,M Type Pyruvate Kinase,M1 Type Pyruvate Kinase,M2 Type Pyruvate Kinase,Pyruvate Kinase, L-Type,Pyruvate Kinase, M-Type,Pyruvate Kinase, M1-Type,Pyruvate Kinase, M2-Type,Pyruvate Kinase, R-Type,R Type Pyruvate Kinase
D004229	Dithiothreitol	A reagent commonly used in biochemical studies as a protective agent to prevent the oxidation of SH (thiol) groups and for reducing disulphides to dithiols.	Cleland Reagent,Cleland's Reagent,Sputolysin,Clelands Reagent,Reagent, Cleland,Reagent, Cleland's
D004789	Enzyme Activation	Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme.	Activation, Enzyme,Activations, Enzyme,Enzyme Activations
D006593	Hexokinase	An enzyme that catalyzes the conversion of ATP and a D-hexose to ADP and a D-hexose 6-phosphate. D-Glucose, D-mannose, D-fructose, sorbitol, and D-glucosamine can act as acceptors; ITP and dATP can act as donors. The liver isoenzyme has sometimes been called glucokinase. (From Enzyme Nomenclature, 1992) EC 2.7.1.1.	Hexokinase A,Hexokinase D,Hexokinase II
D000254	Sodium-Potassium-Exchanging ATPase	An enzyme that catalyzes the active transport system of sodium and potassium ions across the cell wall. Sodium and potassium ions are closely coupled with membrane ATPase which undergoes phosphorylation and dephosphorylation, thereby providing energy for transport of these ions against concentration gradients.	ATPase, Sodium, Potassium,Adenosinetriphosphatase, Sodium, Potassium,Na(+)-K(+)-Exchanging ATPase,Na(+)-K(+)-Transporting ATPase,Potassium Pump,Sodium Pump,Sodium, Potassium ATPase,Sodium, Potassium Adenosinetriphosphatase,Sodium-Potassium Pump,Adenosine Triphosphatase, Sodium, Potassium,Na(+) K(+)-Transporting ATPase,Sodium, Potassium Adenosine Triphosphatase,ATPase Sodium, Potassium,ATPase, Sodium-Potassium-Exchanging,Adenosinetriphosphatase Sodium, Potassium,Pump, Potassium,Pump, Sodium,Pump, Sodium-Potassium,Sodium Potassium Exchanging ATPase,Sodium Potassium Pump
D000255	Adenosine Triphosphate	An adenine nucleotide containing three phosphate groups esterified to the sugar moiety. In addition to its crucial roles in metabolism adenosine triphosphate is a neurotransmitter.	ATP,Adenosine Triphosphate, Calcium Salt,Adenosine Triphosphate, Chromium Salt,Adenosine Triphosphate, Magnesium Salt,Adenosine Triphosphate, Manganese Salt,Adenylpyrophosphate,CaATP,CrATP,Manganese Adenosine Triphosphate,MgATP,MnATP,ATP-MgCl2,Adenosine Triphosphate, Chromium Ammonium Salt,Adenosine Triphosphate, Magnesium Chloride,Atriphos,Chromium Adenosine Triphosphate,Cr(H2O)4 ATP,Magnesium Adenosine Triphosphate,Striadyne,ATP MgCl2
D001345	Autoradiography	The making of a radiograph of an object or tissue by recording on a photographic plate the radiation emitted by radioactive material within the object. (Dorland, 27th ed)	Radioautography
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D012441	Saccharomyces cerevisiae	A species of the genus SACCHAROMYCES, family Saccharomycetaceae, order Saccharomycetales, known as "baker's" or "brewer's" yeast. The dried form is used as a dietary supplement.	Baker's Yeast,Brewer's Yeast,Candida robusta,S. cerevisiae,Saccharomyces capensis,Saccharomyces italicus,Saccharomyces oviformis,Saccharomyces uvarum var. melibiosus,Yeast, Baker's,Yeast, Brewer's,Baker Yeast,S cerevisiae,Baker's Yeasts,Yeast, Baker