X‐Ray Investigation of the Binding of 1,10‐Phenanthroline and Imidazole to Horse‐Liver Alcohol Dehydrogenase

Abstract

We have studied the binding of two inhibitor molecules, imidazole and 1 ,lo-phenanthroline, to liver alcohol dehydrogenase by crystallographic methods. X-ray data for the imidazole complex were collected to 0.29-nm resolution and for the 1,lO-phenanthroline complex to 0.45-nm resolution. In both cases we found only one peak in the difference electron density maps close to the active zinc atom. The peak corresponding to 1,lO-phenanthroline overlaps the site of the density of the zinc-bound water in the apoenzyme and the imidazole density partly overlaps this density. We can not discern any additional peaks close to the zinc atom which would correspond to new positions of bound water. We thus conclude that both these inhibitors bind to the catalytic zinc atom and that upon binding they displace the water molecule that is firmly bound to this zinc atom in the apoenzyme. We do not see any structural changes in the remaining part of the molecule.The three-dimensional structure of horse liver alcohol dehydrogenase is known to high resolution [l]. It is thus now possible to try to elucidate the mechanism of action of this enzyme by crystallographic studies of the binding of suitably chosen inhibitor molecules for both coenzyme and substrates. Details of coenzyme binding have been deduced in this way in several studies using both coenzyme analogues [2 -41, which bind isomorphously to the orthorhombic apoenzyme structure, and a combination of NADH and inhibitor [5], which produces triclinic holoenzyme crystals. These studies confirmed earlier observations [6,7] that the zinc atom, which is necessary for catalytic action, is not directly involved in coenzyme binding. We here report studies of complexes with inhibitor molecules that bind directly to the catalytically active zinc atoms and thus in all probability define the binding areas of substrate molecules. Two such complexes have been studied using the inhibitor molecules 1 ,lo-phenanthroline and imidazole.It towards both coenzyme and substrate but a careful analysis [13,14] demonstrated that this kinetic behaviour could be attributed solely to the formation of an inactive binary enzyme . 1,lO-phenanthroline complex. Using double difference spectrophotometry Yonetani [15,16] could show that this large inhibitor molecule competed with the nicotinamide moiety of the coenzyme and demonstrated the existence of a ternary complex between enzyme, ADP-ribose and 1,lO-phenanthroline. Further solution evidence that this inhibitor binds in the substrate and nicotinamide binding areas has been provided by Sigman [17], who has shown competition between binding of the similar inhibitor 2,2-bipyridine and substrate molecules.Imidazole shows a different inhibition pattern. Theorell and McKinley McKee [18,19] have demonstrated the existence not only of a binary enzyme . imidazole complex but also of inactive ternary complexes with both oxidized and reduced coenzyme. Furthermore they have also shown [18] that imidazole abolishes the pH dependence of NAD+ binding, and thus interferes...

The coordination chemistry of the catalytic zinc ion in alcohol dehydrogenase studied by ab initio quantum chemical calculations

“…Spectroscopic studies of cadmium, copper, or cobalt substituted alcohol dehydrogenase in several instances have been taken to provide evidence for the presence of a fivecoordinated catalytic metal site [18][19][20]. The present results indicate that, to the extent that five-coordinate complexes do form with metal-substituted enzyme, this is likely to reflect the disparity in coordination preferences of different metal ions and cannot be taken to suggest that the catalytic metal site in native enzyme is five-coordinate.…”

“…On the other hand, there is also strong crystallographic and spectroscopic evidence showing that binding to zinc of certain bidentate inhibitors is fivecoordinate [18][19][20]. Furthermore, spectroscopic studies of metal-substituted alcohol dehydrogenase have indicated that several binary and ternary complexes may be fivecoordinate [11][12][13][20][21][22].…”

The Protein Conformation of Cd‐Substituted Horse Liver Alcohol Dehydrogenase and its Metal‐Site Coordination Geometry in Binary and Ternary Inhibitor Complexes

“…Furthermore, studies on model complexes have indicated that one of the cysteine sulfurs may be involved in non-covalent interactions with the substrate, and that this interaction causes a change of the S-metal-S angle (Shoner et al, 1995). (Boiwe and BrandCn, 1977) open (this work) closed (Eklund et al, 1982b) n. m. closed (Plapp et al, 1978) closed (Ramaswamy et al, 1994) n. m. open (CedergrenZeppezauer, 1983) closed (unpublished results) closed (Al-Karadaghi et al, 1994) flexible rigid rigid rigid Deprotonation of alcohol is usually assumed to occur prior to hydride transfer to the coenzyme (Pocker et al, 1986;Kvassman and Pettersson 1987). Thereby, the substrate will coordinate as an alkoxide ion and we suggest that the local structural change in the metal coordination geometry actually occurs during catalysis.…”

“…When a binary inhibitor complex is formed, in which the Zn-bound water has been exchanged for another ligand like imidazole, pyrazole or phenanthroline, no large structural changes are observed (Boiwe and BrandCn, 1977;Eklund et al, 1982b). Thus, exchange of the zinc-bound water with other small molecules seems to maintain the protein in the open conformation (orthorhombic crystals, space group C222,).…”

Molecular dynamics simulations of alcohol dehydrogenase with a four‐ or five‐coordinate catalytic zinc ion

“…Crystallographic and spectroscopic data showing that binding to zinc of certain bidentate inhibitors is fivecoordinate 14,15 have been taken as evidence in favor of these suggestions. Furthermore, spectroscopic studies of metal-substituted alcohol dehydrogenase have indicated that several binary and ternary complexes may be five-coordinate [6][7][8][15][16][17][18][19][20] , although other spectroscopic investigations contradict these results [21][22][23][24][25] .…”