Site‐Specific Substituted Cobalt(II) Horse Liver Alcohol Dehydrogenases

Abstract

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1 .I .1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives.In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier.Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 pM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing r61e of the catalytic metal ions.The enzyme immobilised on agarose, with unchanged content of active sites Eur. J . Biochem. 41, 475-4841, was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogeneous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.Ahhreviations. Tes, 2-{ [tris(hydroxymethyl)methyl]amino}etha-nesulfonic acid; Tris: Tris(hydroxymethy1)-aminomethane; Mes, 2-(N-morpholino)ethanesulfonic acid. The differently substituted metallo-alcohol dehydrogenases are named as follows. The metal ions are differentiated as catalytic and noncatalytic by the characters (c) and (n) after the chemical symbol, i.e. Zn(c)zZn(n)~-enzyme = native enzyme; H4Zn(n)z-enzyme = enzyme depleted of the catalytic zinc ions; Zn(c)2Co(n)z-enzyme = enzyme substituted at the noncatalytic binding sites ('green hybrid' enzyme) ; CO(C)ZZn(n)2-enzyme = enzyme ...

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

“…Active-site-specific CoLADH was prepared as described by Maret et aL (14). Its absorbance properties were identical to those described (14), and the extent of replacement of the active-site Zn2' by Co2+ was >85% as estimated by atomic absorption analysis. Preparations ofall enzyme complexes for EPR studies were carried out in a nitrogen atmosphere at 0-40C with use of solvents previously purged with nitrogen.…”

“…* For the crystals used by Plapp and coworkers (10) trations of CoLADH with NAD' and pyrazole and with NADH and isobutyramide show that the equilibrium binding constants for these ligands are comparable with those ofthe native enzyme (14). On this basis, binary and ternary complexes of CoLADH were prepared directly in EPR sample tubes by'addition of appropriate aliquots of NAD' and pyrazole buffered to pH 6.5 with 0.05 M Tes to ensure at least 99% complex formation.…”

Characterisation of a novel cysteine/histidine‐rich metal binding domain from Xenopus nuclear factor XNF7

“…Recently, copper has been shown to bind with high affinity to the normally zinc-binding estrogen receptor DNAbinding domain, using two cysteines per copper atom for ligation. The physiological significance of this observation is however unclear and may be artefactual [34]. In order to monitor any conformational changes that occur upon ligation of metal ion, 1D NMR spectra were collected in the presence and absence of zinc.…”

Binding of ligands to horse liver alcohol dehydrogenase lacking zinc ions at the active sites

“…The apoenzyme was prepared from the holoenzyme by the method of Maret et al [9]. The lyophilized enzyme was dialyzed against 0.02 M sodium phosphate buffer pH 7.2.…”

“…The lyophilized enzyme was dialyzed against 0.02 M sodium phosphate buffer pH 7.2. The concentration of the modified enzyme was determined spectrophotometrically [9] and by titration with NADH [S]. The residual activity of the preparation [lo] revealed the presence of less than 1% of the native enzyme in the apoenzyme.…”