Investigations of the E2 --> E1 conformational change of Na(+),K(+)-ATPase from shark rectal gland and pig kidney via the stopped-flow technique have revealed major differences in the kinetics and mechanisms of the two enzymes. Mammalian kidney Na(+),K(+)-ATPase appears to exist in a diprotomeric (alphabeta)(2) state in the absence of ATP, with protein-protein interactions between the alpha-subunits causing an inhibition of the transition, which occurs as a two-step process: E2:E2 --> E2:E1 --> E1:E1. This is evidenced by a biphasicity in the observed kinetics. Binding of ATP to the E1 or E2 states causes the kinetics to become monophasic and accelerate, which can be explained by an ATP-induced dissociation of the diprotomer into separate alphabeta protomers and relief of the preexisting inhibition. In the case of enzyme from shark rectal gland, the observed kinetics are monophasic at all ATP concentrations, indicating a monoprotomeric enzyme; however, an acceleration of the E2 --> E1 transition by ATP still occurs, to a maximum rate constant of 182 (+/- 6) s(-1). This indicates that ATP has two separate mechanisms whereby it accelerates the E2 --> E1 transition of Na(+),K(+)-ATPase alphabeta protomers and (alphabeta)(2) diprotomers.

The interaction of ATP with the phosphoenzyme of Na(+),K(+)-ATPase from pig kidney, rabbit kidney, and shark rectal gland was investigated using the voltage-sensitive fluorescent probe RH421. In each case, ATP concentrations >or=100 microM caused a drop in fluorescence intensity, which, because RH421 is sensitive to the formation of enzyme in the E2P state, can be attributed to ATP binding to the E2P phosphoenzyme. Simulations of the experimental behavior using kinetic models based on either a monomeric or a dimeric enzyme mechanism yielded a K(d) for ATP binding in the range 140-500 muM. Steady-state activity measurements and independent measurements of the phosphoenzyme level via a radioactive assay indicated that ATP binding to E2P causes a deceleration in its dephosphorylation when acting in the Na(+)-ATPase mode, i.e., in the absence of K(+) ions. Both the ATP-induced drop in RH421 fluorescence and the effect on the dephosphorylation reaction could be attributed to an inhibition of dissociation from the E2P(Na(+))(3) state of the one Na(+) ion necessary to allow dephosphorylation. Stopped-flow studies on the shark enzyme indicated that the ATP-induced inhibition of dephosphorylation is abolished in the presence of 1 mM KCl. A possible physiological role of allosteric binding of ATP to the phosphoenzyme could be to stabilize the E2P state and stop the enzyme running backward, which would cause dissipation of the Na(+) electrochemical potential gradient and the resynthesis of ATP from ADP. ATP binding to E2P could also fix ATP within the enzyme ready to phosphorylate it in the subsequent turnover.

2‐Cyanoacetamido‐4‐methylthiazole (1) was utilized as a versatile precursor for the construction of new thiazole clubbed thiazolidine, thiophene, pyridine, or chromene scaffold. The base‐catalyzed addition of 1 to phenyl isothiocyanate followed by subsequent treatment of the produced thiocarbamoyl intermediate with ethyl chloroacetate or chloroacetonitrile furnished the corresponding thiazolyl‐thiazolidine and thiazolyl‐thiophene hybrids. The reactions of compound 1 with chemical reagents, namely, acetylacetone, malononitrile, and/or 2‐(4‐anisylidene)‐malononitrile have been studied and furnished the corresponding thiazole‐pyridine hydrides 8–10. Furthermore, treatment of the precursor 1 with salicylaldehyde, various aryl diazonium chlorides, and/or aromatic aldehydes afforded their corresponding thiazolyl‐chromene hybrid 12, arylhydrazono‐nitriles 13, and unsaturated nitriles 14, respectively. The cytotoxicity of the synthesized compounds was screened against the cell lines HepG2, HCT‐116, and MCF‐7. Compounds 8, 10, and 12 recorded the best results, which was illustrated by molecular docking. Molecular Operating Environment molecular docking calculations carried out here is to rationalize correlation between docking results and biological data of thymidylate synthase (Protein Data Bank code: IHVY) inhibition. Docking has been carried out in the same co‐crystallographic inhibitor binding site to predict if the binding mode of active compounds is analogous to that of native inhibitor.

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