Relation between conformations and activities of lipoamide dehydrogenase: I. Relation between diaphorase and lipoamide dehydrogenase activities upon binding of fad by the apoenzyme

Abstract

1. 1. The apoenzyme of lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) can be prepared by acid (NH 4) 2SO 4 treatment. This apoenzyme is able to bind 1 mole FAD per 48 000 g protein. For this process ΔH=−8300 cal-mol −1 and ΔS=−4 e.u. The activities obtained are dependent on the temperature employed. At 0–5°, no return of the activity with lipoate is observed but the activity with 2,6-dichlorophenol indophenol (DCIP) is increased to a level 20-fold higher than that of the holoenzyme. The results indicate that at least two different conformations are involved in this temperature range. At 5–25° the activity with lipoate returns in a bimolecular reaction with respect to protein concentration, at the expense of the activity with DCIP. The activation energy is 21 000 cal·mole −1. 2. 2. The apoenzyme of the enzyme modified by Cu 2+ prepared in a similar way is also able to bind FAD. However in this case, the DCIP activity returns to its original level. Treatment of the normal apoenzyme with Cu 2+ leads to a total loss of the FAD-binding capacity. 3. 3. Freezing and thawing at low ionic strength leads to an enzyme which shows stimulated activity with DCIP and diminished activity with lipoate; bovine serum albumin. (NH 4) 2SO 4 and EDTA protect against these changes. Incubation at 25° reverses this modification. Freezing and thawing induces a reversible shift in the flavin absorption from 455 mμ to 452 mμ; the maximum of the fluorescence emission shifts 2 mμ to the blue side of the spectrum.