Glucose 6-phosphate dehydrogenase from brewers' yeast (Zwischenferment). Further observations on the ligand-induced macromolecular association phenomenon: kinetic properties of the two-chain protein species; and studies on the enzyme-substrate interactions.

Abstract In the presence of EDTA3−, the two-subunit species of apoenzyme (Mr 102,000 g/mole) ( Yue, Noltmann, and Kuby (1967) Biochemistry6, 1174) maintains its integrity without further dissociation into component subunits over a broad range of pH values to 9.8 and in very dilute solutions, as demonstrated by sedimentation equilibrium studies. In the absence of EDTA3−, studies on the ligand-induced macromolecular association to the four-subunit species ( Yue, Noltmann, and Kuby (1969) J. Biol. Chem.244, 1353–1364) have been further extended for NADP+ to include ultracentrifugal titrations at pH 7.8 and 3 °C. A significant concentration of EDTA3−, as before, will inhibit these macromolecular association reactions and stabilize the apoenzyme species in solution. NADPH is similar to NADP+ in that it will induce the formation of the four-subunit species; but by way of contrast, glucose 6-phosphate2−will not. In fact, the addition of glucose 6-phosphate2− to NADPH solutions may actually inhibit or reverse the association reaction induced by NADPH, and in this respect, glucose 6-phosphate2− acts in a fashion not unlike the EDTA− ion. Thus, the ratio of ( NADP ) 0 ( Glc -6-P) 0 as well as their absolute concentrations will affect the extent of dimerization of the enzyme in the absence of any other modifiers. The use of EDTA3− has now permitted direct steady-state kinetic measurements on the two-chain species alone, which has proved to be active even in the presence of high concentrations of EDTA3−. The steady-state kinetic mechanism of the “monomeric” enzyme species appears to have reduced to a relatively simple case, viz., to that of a random quasi-equilibrium type with a rate-limiting step at the interconversion of the ternary complexes and with independent binding of the substrates; values for the kinetic parameters have been derived for this mechanism. The product inhibition pattern of NADPH (competitive with respect to both substrates) would point, in addition, to the absence of any kinetically significant concentrations of dead-end complexes with the two-chain species. To confirm the assigned kinetic mechanism for the two-chain enzyme, measurements of the equilibrium binding of the substrates were undertaken by several techniques. By gel filtration through Sephadex G-25, in the presence of EDTA3−, two equilavent binding sites for NADP+ per mole of apoenzyme (or one per subunit)- is observed, with a value for the intrinsic dissociation constant approximating the kinetically determined value. On the other hand, in the absence of EDTA3−, the nonlinear Scatchard plot obtained is likely a reflection of the superimposed association-dissociation reactions involving the protein. Similar quantitative studies for the ligand NADP+, in the presence and absence of EDTA3−, were conducted by equilibrium dialysis, by a uv difference-spectral technique, and by measurement of the protection afforded by either substrate against inactivation by 5,5′-dithiobis(2-nitrobenzoic acid), which apparently reacts with only a single exposed sulfhydryl group per subunit. Coincident with these studies, a total amino acid composition for this protein is reported. In the case of glucose 6-phosphate, by the technique of equilibrium dialysis, there appears to be two major equivalent binding sites (or one per subunit) with an intrinsic dissociation constant approximating that measured kinetically, as well as two additional but very weak sites (or one additional site per subunit). Moreover, EDTA3− exerts no effect on the equilibrium binding behavior of Glc-6-P, in agreement with the observation that Glc6-P does not induce the formation of the tetra-chain species. Glucose 6-phosphate will also protect the enzyme against inactivation by 5,5′dithiobis(2-nitrobenzoic acid), pointing to a close proximity of both binding sites for Glc-6-P and NADP+ to the single exposed sulfhydryl group per subunit. Finally, fluorometric observations have qualitatively confirmed the existence of both NADPH- and NADP+- enzyme binary compounds.