Interaction of Coenzyme with Differently Prepared Zinc-free (Apo) Horse Liver Alcohol Dehydrogenases
C. W. Hoagstrom 1, I. Iweibo 1, and H. Weiner 1
From the
1 From the Department of Biochemistry, Purdue University, Lafayette, Indiana 47907
The 4 zinc atoms in horse liver alcohol dehydrogenase (EC 1.1.1.1) have been removed three different ways: dialysis against ethylenediaminetetraacetic acid at pH 6.0; dialysis against acetate at pH 5.0, and dialysis against succinate at pH 5.0. The properties of the three apoenzymes differ. Only the apoenzyme prepared by the EDTA treatment will interact with the antibodies produced by native alcohol dehydrogenase, which contains 4 zinc atoms per mole. Also, only the EDTA-prepared apoenzyme will bind NADH.
The apoenzymes are catalytically inactive, but since the enzyme produced by EDTA treatment still binds coenzyme and interacts with antibody for native enzyme, it can be assumed that the gross conformation of this apoenzyme is similar to that of native alcohol dehydrogenase. The reason for the inactivity of the enzyme may be associated with either substrate binding or activation of the substrate.
The binding of NADH to the EDTA-prepared apoenzyme was studied by polarization of fluorescence. It was found that two nonidentical NADH-binding sites remained in the EDTA-prepared apoenzyme, Kd = 0.27 ± 0.01 µm and 1.44 ± 0.24 µm. The site with high affinity has the same Kd as that of native enzyme.
Submitted on July 7, 1969
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