Crystal structures of the active site in specifically metal-depleted and cobalt-substituted horse liver alcohol dehydrogenase derivatives.

Two derivatives of horse liver alcohol dehydrogenase (LADH) in which the active site is specifically metal-depleted [H4Zn(n)2LADH] or specifically Co-substituted [Co(c)2-Zn(n)2LADH] have been studied by crystallographic methods. (In these formulae, "n" identifies the noncatalytic zinc ion and "c" identifies the catalytic metal ion.) X-ray data were collected for H4Zn(n)2LADH to 2.7-A resolution and for Co(c)2Zn(n)2LADH to 2.4-A resolution. Difference Fourier maps demonstrate clearly that the catalytic zinc ions are removed in H4Zn(n)2LADH, whereas the noncatalytic zinc ions are still present. A 2.5-A shift in the sulphur position of cysteine-46 and a slight torsion of the imidazole ring of histidine-67 are the only changes in the protein structure that could be detected when compared to the native zinc enzyme. The structure of Co(c)2Zn(n)2LADH is essentially the same as that of the native enzyme. Each cobalt ion is bound to the ligands cysteine-46, cysteine-174, and histidine-67 and to a water molecule in a distorted tetrahedral geometry. A slight change in the position of histidine-67 was found. No further structural changes could be observed in the protein.