Crystal structure determinations of coenzyme analogue and substrate complexes of liver alcohol dehydrogenase: binding of 1,4,5,6-tetrahydronicotinamide adenine dinucleotide and trans-4- (N,N-dimethylamino) cinnamaldehyde to the enzyme
Cedergren-Zeppezauer, E.; Samama, J.P.; Eklund, H.
Biochemistry 21(20): 4895-4908
1,4,5,6-Tetrahydronicotinamide adenine dinucleotide (H2NADH) was used as a reduced coenzyme analog to study a simulated transient intermediate of [horse] liver alcohol dehydrogenase (LADH) with trans-4-(N,N-dimethylamino)cinnamaldehyde (DACA) as a substrate. X-ray diffraction data of 2 crystal modifications were analyzed to 2.9-.ANG. resolution: an orthorhombic complex where the coenzyme analog binds to the apoenzyme conformation and a triclinic complex with coenzyme and substrate bound to the holoenzyme conformation. The interpretation of difference electron density maps for both crystal modifications is based on calculations where the phase angles were derived from refined enzyme models. H2NADH binds to the coenzyme binding domain in an extended conformation. The tetrahydronicotinamide ring is situated within the active site region in both enzyme conformations, but the orientation of the ring plane in relation to the active site Zn atom is very different. In the triclinic LADH-H2NADH-aldehyde complex the coenzyme analog binds in a similar way as a NADH to a ternary inhibited complex. The combined effect of the presence of H2NADH and the large DACA substrate triggers the gross conformational change of the protein, which involves the movement of the catalytic domains in the dimeric molecule. In contrast, only local structural changes in the orthorhombic LADH-H2NADH complex are observed upon coenzyme analog binding. A network of H-bonds between water molecules, the coenzyme, and side chains from the catalytic domain bridges the cleft between the domains in the orthorhombic complex. Since the conformational change narrows the cleft, no such water arrangement is found in the triclinic substrate complex. In the orthorhombic complex the substrate binding pocket is occupied by a methylpentanediol (MPD) molecule used as the precipitating agent during crystallization. MPD binds within H-bonding distance to Zn-bound water. The X-ray analysis shows that the aldehyde molecule is directly liganded to the metal atom in the triclinic LADH-H2NADH-DACA complex. The binding of the chromophoric aldehyde substrate within the single crystals used for diffraction experiments was monitored by microspectrophotometric measurements.