The protein conformation of Cd-substituted horse liver alcohol dehydrogenase and its metal-site coordination geometry in binary and ternary inhibitor complexes.
The coordination geometry of the metal at the active site in Cd-substituted horse liver alcohol dehydrogenase (LADH) has been investigated for the binary complexes of LADH with imidazole, isobutyramide, decanoic acid and Cl-, and for the ternary complexes of LADH with NADH and imidazole, NADH and isobutyramide, NAD+ and decanoic acid and NAD+ and Cl-, by using the method of perturbed angular correlation of gamma-rays (PAC). The spectral results are consistent with a flexible structure around the metal for the binary complexes with inhibitors. For ternary complexes, however, a rigid structure is observed. An exception is the ternary complex between LADH, NADH and imidazole, in which the metal site is still flexible. Comparing with available structures determined by X-ray crystallography, we found a correlation between open structures and flexible metal sites, and between closed structures and rigid metal sites. This indicates that the PAC technique can be applied to distinguish the two conformations in solution. The spectral parameters, omega(o) and eta, of the experiments, except for the complexes with imidazole, fall into two groups: one with low omega(o) and one with high omega(o) (eta is relatively constant in all experiments). In this work it is clarified that the low omega(o) values are connected with the presence of a negatively charged solvent ligand. Using an angular-overlap approach to interpret the results, the low omega(o) values are found to be compatible with a coordination geometry where the S-Cd-S (Cys174 and Cys46 coordinate to the metal) angle is about 110 degrees as suggested in [Hemmingsen, L., Bauer, R., Danielsen, E., Bjerrum. M. J., Zeppezauer, M., Adolph, H. W., Formicka, G. & Cedergren-Zeppezauer, E. (1995) Biochemistry 34, 7145-7153], whereas high omega(o) values are compatible with an S-Cd-S angle of 130 degrees. The presence of a negatively charged metal ligand, therefore, might trigger the movement of the sulfur of Cys174. As it is believed that alcohols coordinate to the metal as alcoholate ions this could be important for catalysis.
Department of Mathematics and Physics, Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
L
L
Hemmingsen
R
R
Bauer
M J
MJ
Bjerrum
H W
HW
Adolph
M
M
Zeppezauer
E
E
Cedergren-Zeppezauer
D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000577:Amides / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry; D018360:Crystallography, X-Ray; D003652:Decanoic Acids / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D006736:Horses; D007093:Imidazoles / Q000737:chemistry; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D015394:Molecular Structure; D009243:NAD / Q000737:chemistry; D011487:Protein Conformation
D000577:Amides; D002712:Chlorides; D003652:Decanoic Acids; D004791:Enzyme Inhibitors; D007093:Imidazoles; D046911:Macromolecular Substances; D002104:Cadmium; D009243:NAD; C031071:decanoic acid; C029899:imidazole; C030197:isobutyramide; D000426:Alcohol Dehydrogenase