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-TETRAHYDRONICOTINAMIDE-ADENINE DINUCLEOTID

Horse liver alcohol dehydrogenase (EC 1.1.1.1), 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, 4882–4892]. 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 ran 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 H2NADH), 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. Copyright © 1979, Wiley Blackwell. All rights reserved