113Cd NMR in binary and ternary complexes of cadmium-substituted horse liver alcohol dehydrogenase.
The 113Cd NMR has been observed for Cd(II)-substituted horse liver alcohol dehydrogenase (LADH) and its complexes with coenzymes and several substrate analogs. Compared to free enzyme, the catalytic Cd(II) resonance is shielded by 41--41 ppm in both LADH-NADH and LADH-NAD+. In ternary complexes of LADH-NAD+ with either trifluoroethanol or pyrazole, this resonance narrows and is deshielded by 75 ppm. The LADH-NADH-butyramide complex gives only 3 ppm of deshielding relative to the LADH-NADH resonance. At pH = 10.3, the catalytic resonance of unbound LADH is broadened and slightly deshielded. No other resonances are dependent upon pH in the range 8--10. These data are the most consistent with a second sphere coordination of the substrate analogs to the catalytic metal ion. The observed difference between complexes of the alcohol analogs and the aldehyde analog would then be explained as the presence of a hydroxide versus a water molecule, respectively, in the first coordination sphere. The data also show that the pKa of the coordinated water on the Zn(II) in the native LADH is close to 9.2 as previously assumed, whereas the pKa of the Zn(II)-bound water in the LADH-NAD+ complex is most likely greater than 9 and not 7.6 as previously assumed.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D002104:Cadmium / Q000494:pharmacology*; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D009243:NAD; D010084:Oxidation-Reduction; D011485:Protein Binding
D046911:Macromolecular Substances; D002104:Cadmium; D009243:NAD; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase