Measurements of binding of certain divalent cations to yeast apoenolase were made using a pH-meter, chromatography, a divalent cation electrode, and ultrafiltration. The binding of the activating metal ions Mg2+ and Co2+ and the nonactivator Ca2+ were studied as functions of the presence or absence of substrate/product, phosphate, and fluoride or level of Tb3+. The data suggest phosphate and fluoride increase Mg2+ binding but not Ca2+ binding. Substrate/product appears to increase Ca2+ binding as well as that of Mg2+ and Co2+. In the presence of substrate, Co2+ binding was 5-6 mol/mol dimer. In the absence of substrate/product, Tb3+ reduced Co2+ binding from 4 mol/mol to 2. These data are interpreted in terms of binding to "conformational," "catalytic" (substrate/product dependent), and "inhibitory" sites. Measurements of Tb3+ fluorescence quenching by Co2+ suggested that the distance between "conformational" sites on the two subunits was large, while the distance between "conformational" and "inhibitory" sites was ca. 17 +/- 4 A. Potentiometric titrations of apoenzyme with Ca2+ and Mg2+ showed that the metal ions produced the same proton release in the presence or absence of substrate/product. If phosphate and fluoride were present, then more protons were released if Ca2+ was the titrant rather than Mg2+, suggesting a difference in ionization state in the complex with the activating metal. Electron paramagnetic resonance studies of Co2+ binding to the various sites in the enzyme are presented. The Co2+ bound to all three sites appears to be high spin, consistent with a preponderance of oxyligands in an octahedral environment. Substrate, citrate, and a strongly binding substrate analogue strongly enhance the hyperfine structure of conformational Co2+. This is interpreted as the result of a change in interaction of an axial ligand to conformational Co2+ produced by carbon-3 of substrate or analogue.

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Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Brewer JM, Carreira LA, Collins KM, Duvall MC, Cohen C, DerVartanian DV

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