The binding of 1,10-phenanthroline to specifically active-site cobalt(II)-substituted horse-liver alcohol dehydrogenase. A probe for the open-enzyme conformation.
We have studied the binding of 1,10-phenanthroline to specifically active-site cobalt(II)-substituted horse-liver alcohol dehydrogenase [Co(II)-LADH]. The dissociation constant is a factor of 6500 smaller than in the native enzyme. Spectral evidence is given which shows that 1,10-phenanthroline does not remove the catalytic Co(II) ion and that binding of 1,10-phenanthroline renders the catalytic metal ion pentacoordinate. The maximum limiting rate constant for the association of 1,10-phenanthroline to Co(II)-LADH is about 60 s-1. This is about a third of the value (169 s-1) determined for native horse-liver alcohol dehydrogenase, Zn(II)LADH [Frolich et al. (1978) Arch. Biochem. Biophys. 189, 471-480]. For cadmium(II)-substituted horse-liver alcohol dehydrogenase, [Cd(II)LADH] the maximum limiting rate constant for association of 1,10-phenanthroline increased to 590 s-1. These findings demonstrate that the rate-limiting step is strongly dependent on the chemical nature of the catalytic metal ion and its immediate environment. 1,10-Phenanthroline is shown to bind to the Co(II)-LADH.NAD+ complex in the open conformation. The maximum limiting rate constant remains unchanged in the presence of NAD+. The data have been used to derive a kinetic scheme for the formation of ternary complexes including NAD+ that involves a slow intermediary step.
Sartorius
C
C
Fachrichtung Biochemie, Universität des Saarlandes, Saarbrücken, Federal Republic of Germany.
Dunn
M F
MF
Zeppezauer
M
M
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002614:Chelating Agents / Q000378:metabolism*; D003035:Cobalt / Q000494:pharmacology*; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010618:Phenanthrolines / Q000378:metabolism*; D011487:Protein Conformation; D011720:Pyrazoles / Q000378:metabolism; D013053:Spectrophotometry
D002614:Chelating Agents; D010618:Phenanthrolines; D011720:Pyrazoles; D009243:NAD; D003035:Cobalt; C031280:pyrazole; D000426:Alcohol Dehydrogenase; C025205:1,10-phenanthroline