| Abstract: | The CD of GDH–NADH complexes was measured in order to reexamine the binding of coenzyme to GDH. The existence of two distinct Cotton effects associated with two separate NADH binding sites/subunit was confirmed with native, polymerizing and crosslinked, unpolymerizing enzyme. CD titration of the high-affinity NADH sites revealed significant dependence of the optical activity of the bound coenzyme on the state of protein association. Molar ellipticity of bound NADH decreased with the increasing degree of polymerization of GDH. It is suggested that the high-affinity NADH sites are loacted at or near the association interface. Binding of NADH to the low-affinity sites, in the presence of GTP, leads to an inversion of the CD spectrum of GDH–NADH complexes. This inversion is not related to the polymerization of GDH. However, for proper analysis of the CD of NADH bound to the low-affinity sites, a correction for the effect of polymerization on the optical activity of NADH bound to the high-affinity sites is required. |
