Interaction of extrinic fluoresence probes with E. coli glutamine synthetase

Abstract

Binding of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) to adenylylated (E--11) glutamine synthetase is cooperative and time-dependent, with 3 dye sites per subunit. In fluorescence polarization experiments TNS and pyrene butyrate give normalized Perrin plots that indicate a symmetrical arrangement of dye excited state dipoles, relative to the rotational axis of the oblate ellipsoid of the dodecameric native enzyme.

Catalytic cooperativity and subunit interactions in E. coli glutamine synthetase binding and kinetics with methionine sulfoximine and related inhibitors

“…with Met(0)(NH) + enzyme-ATP show saturation of rates as a function of [MSOX] that gives /zH < 1.0. (i) Biphasic binding of the fluorescent dye TNS to the enzyme can be interpreted as negative cooperativity (Wedler et al, 1977) since only one TNS is bound maximally per subunit.3 (j) Both the present results (Figure 2) and those of Meek & Villafranca (1980) indicate apparent negative cooperativity in the kinetics of binding of ammonia to the enzyme.…”

“…The relation of these effects to enzyme catalysis will be examined in the following sections. It is noteworthy at this point that the binding of the fluorescent dye TNS [2-(p-toluidinyl)naphthalene-6-sulfonate] to E. coli GS also exhibited biphasic saturation (Wedler et al, 1977) and that subsequent exper-iments3 have revealed that only one TNS binds per subunit.…”