Active-Site-Specific Reconstituted Cobalt(II) Horse-Liver Alcohol Dehydrogenase. Changes of the Spectra of the Substrate trans-4-(N,N-Dimethylamino)-cinnamaldehyde and of the Catalytic Cobalt Ion upon Ternary Complex Formation with NADH and 1,4,5,6-Tetrahydronkotinamide-Adenine Dinucleotide

Abstract

Horse liver alcohol dehydrogenase (EC 1 .l. 1. l), specifically reconstituted with cobaltous ions in the catalytic metal-binding sites, forms ternary complexes with the chromophoric substrate trans-4-(N,N-dimethylamino)-cinnamaldehyde and NADH or 1,4,5,6-tetrahydronicotinamide-adenine dinucleotide (H2NADH), in close analogy to the native zinc enzyme [Dunn, M. F. and Hutchison, J. S. (1973) Biochemistry, 12, 4882-48921. Control experiments with enzyme depleted of metal in the catalytic sites demonstrate that presence of a metal ion is an absolute requirement for binding of the substrate. The spectra of both the chromophore and the metal ion are changed significantly upon complex formation. The red shift of the visible absorption band of the substrate molecule is 14 nm larger for both the complexes with NADH and H2NADH as compared to the native zinc enzyme; we take this as evidence for direct coordination of the substrate's carbonyl oxygen to the catalytic cobalt ion. The greatly increased intensity of the metal's visible d-d band upon substrate binding adds support to this conclusion. Since binding of the coenzyme as well as of the analog, which have to precede the binding of the substrate, leads to considerable red shifts of the d-d band at 650 nm (28 nm for NADH and 21 nm for HzNADH), a distortion of the coordination sphere of the catalytic metal ion is triggered prior to substrate binding. Presumably the coenzyme-triggered conformation change of the protein involving the catalytic metal ion results in a modulation of the Lewis acid strength of the metal which then is able to coordinate and activate the substrate.It is well established that two of the four zinc ions in horse liver alcohol dehydrogenase participate directly in the reaction catalyzed by this enzyme [l]. However, the detailed mode of participation of metal ions in the catalytic cycle is unclear and still a matter of debate [2]; apparently the individual steps in this catalysis depend on a subtle interplay between substrate, coenzyme, metal and protein structure. In order to elucidate the chemical nature and the time course of these interdependent events, molecular probes have been introduced into the system, such asAbbreviations. Chromophore, trans-4-(iV,N-dimethylamino)-cinnamaldehyde; H2NADH, 1,4,5,6-tetrahydronicotinamideadenine dinucleotide; in Me(c)Me(n)-enzyme, Me = metal, c = catalytic, n = noncatalytic, enzyme = horse liver alcohol dehydrogenase.Enzyme. Horse liver alcohol dehydrogenase (EC 1.1.1.1).cobaltous ions instead of the native zinc [3,4] and chromophoric substrates [5 -71. In particular, the chromophore trans-4-(N,N-dimethylamino)-cinnamaldehyde serves the dual purpose of being a substrate at neutral pH and forming a stable transient in the complex with the enzyme and NADH at high pH [ 5 ] . In this stable ternary complex, the absorption maximum of the chromophore is redshifted by 66nm. This has been taken as strong evidence for a direct coordination of the substrate's carbonyl group with the catalytic zinc ion, which acts as a Le...

Thermoanaerobacter brockii alcohol dehydrogenase: Characterization of the active site metal and its ligand amino acids

“…When the catalytic zinc of TBADH was substituted by such metals as cobalt or cadmium, the rates of the enzymatic reactions were 1.3 and 0.02 of those for the zinc enzyme. Similar results have been reported for HLADH (Dunn & Hutchison, 1973;Dietrich et al, 1979;Moms et al, 1980;Dunn et al, 1982). The results correlated with the expected order of Lewis acidity (for a tetrahedral ligand field) Co 2 Zn >> Cd (Dunn et al, 1982).…”

Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis

“…45 Spectra showed that trans -4-( N , N -dimethylamino)cinnamaldehyde binds to the Co(II) in the complex with 1,4,5,6-tetrahydronicotinamide adenine dinucleotide. 81,82 The NMR study was extended to ternary complexes with NADH and dimethyl sulfoxide or NAD + and 2,2,2-trifluoroethanol, and again it was concluded that the oxygens of the ligands were not directly ligated to the metal. 83 However, other studies concluded that the paramagnetic effects of Co(II) on the relaxation of water or alcohol protons were too small to give reliable determinations of the distance of the protons from the metal.…”

“…However, a low-resolution X-ray crystallography study suggested that substrates and inhibitors bind directly to the zinc in enzyme–coenzyme complexes that resemble Michaelis complexes . Spectra showed that trans -4-( N , N -dimethylamino)­cinnamaldehyde binds to the Co­(II) in the complex with 1,4,5,6-tetrahydronicotinamide adenine dinucleotide. , The NMR study was extended to ternary complexes with NADH and dimethyl sulfoxide or NAD + and 2,2,2-trifluoroethanol, and again it was concluded that the oxygens of the ligands were not directly ligated to the metal . However, other studies concluded that the paramagnetic effects of Co­(II) on the relaxation of water or alcohol protons were too small to give reliable determinations of the distance of the protons from the metal. , After higher-resolution X-ray studies clearly showed that the oxygens of the ligands are directly ligated to a tetracoordinated zinc and that there is no water near the zinc, we proposed that the oxygen of the substrate is bound to the zinc, and the proton is transferred to or from the substrate through a hydrogen-bonded system, including the hydroxyl group of Ser-48 and the imidazole group of His-51 …”

Magnetic and optical properties of copper-substituted alcohol dehydrogenase: a bisthiolate copper (II) complex

“…X-ray crystallography of the metal-substituted cadmium and cobalt enzymes has shown that the distorted tetrahedral coordination geometry is retained [22,23]. Spectroscopic studies with Co(II)-and Cd(II)-substituted enzymes [24][25][26] have suggested geometrical changes at the catalytic metal site upon coenzyme binding similar to those observed with the zinc enzyme. The structure of the copper-substituted complex of HLADH with DMSO has been determined to 2.10 A H [27].…”