Cobalt(II)-substituted class III alcohol and sorbitol dehydrogenases from human liver
- 1 December 1989
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 28 (26), 9944-9949
- https://doi.org/10.1021/bi00452a011
Abstract
The catalytic zinc atoms in class III (.chi.) alcohol dehydrogenase (ADH) and sorbitol dehydrogenase (SDH) from human liver has been specifically removed and replaced by cobalt(II) with a new ultrafiltration technique. The electronic absorption spectrum of class III cobalt ADH (.epsilon.638 = 870 M-1 cm-1) is nearly identical with those of active site substituted horse EE and human class I (.beta.1.beta.1) cobalt ADH. Thus, the coordination environment of the catalytic metal is strictly conserved in these enzymes. However, significant differences are noted when the spectra of class III ADH .cntdot. coenzyme complexes are compared to the corresponding spectra of the horse enzyme. The spectrum of class III ADH .cntdot. NADH is split into three bands, centered at 680, 638, and 562 nm. The class III ADH .cntdot. NAD+ species resembles the alkaline form of the corresponding horse enzyme complex but without exhibiting the pH dependence of the latter. These spectral changes underscore the role of the coenzymes in differentially fine tuning the catalytic metal for its particular function in each ADH. The noncatalytic zinc of class III ADH exchanges with cobalt at pH 7.0. While 9 residues out of 15 in the loop surrounding the noncatalytic zinc of class III ADH differ from those of the class I ADH, the electronic absorption spectra of cobalt in the noncatalytic metal site of class III ADH establish that the coordination environment of this site is conserved as well. The spectrum of cobalt SDH differs significantly from those of cobalt ADHs. Comparison of absorption maxima corroborates the recent proposal [Eklund, H., Horjales, E., Jornvall, H., Branden, C.-I., and Jeffery, J. (1985) Biochemistry 24, 8005-8012] that in SDH only one cysteine is a metal ligand. The relatively low molar absorptivity of cobalt SDH (.epsilon.562 = 230 M-1 cm-1) indicates a distorted tetrahedral or pentacoordinate structure of the metal ion. Binding of NADH to cobalt SDH induces a transition to a tetrahedral metal site as manifested by a hyperchromic effect on the main absorption band (.epsilon.590 = 410 M-1 cm-1). This perturbation is unique when compared to cobalt ADHs and represents yet another type of coenzyme-induced modulation of the catalytic metal in this family of enzymes.This publication has 19 references indexed in Scilit:
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