Dissociation of outer-sphere water is rate-limiting for the binding of ligands in the active site of horse liver alcohol dehydrogenase.
The association of imidazole and auramine O to native horse-liver alcohol dehydrogenase [Zn(II)LADH] and active-site specifically cobalt(II)-substituted horse-liver alcohol dehydrogenase [Co(II)LADH], respectively, has been investigated. In all cases [except imidazole binding to Zn(II)LADH in the presence of auramine O] the association rates approached an upper limit (kmax). The kmax values were compared for the metal ligands imidazole (monodentate), 1,10-phenanthroline and 2,2'-bipyridine (bidentate; see also the preceding paper), and for auramine O which does not coordinate to the catalytic metal ion. Independent of the large differences in their structure and metal-bonding capability, all these compounds exhibit common, maximum, limiting rate constants of about 60 s-1 and 200 s-1 for Co(II)LADH and Zn(II)LADH, respectively. These results demonstrate that kmax is strongly dependent on the catalytic metal ion but not on the ligand. The absence of spectral changes in the d-d transitions of the catalytic Co(II) ion upon auramine O binding to Co(II)LADH indicates that the rate-limiting step is not accompanied by a major conformational change. Finally, it is concluded that reactions in the inner coordination sphere of the catalytic metal ion (i.e. the metal-bound water molecule) are not responsible for the step characterized by kmax. We propose the rate-limiting step to consist of the dissociation of one or several water molecules from the second coordination sphere of the catalytic metal ion in the active site of LADH in its open conformation.
Sartorius
C
C
Fachrichtung Biochemie, Universität des Saarlandes, Saarbrücken, Federal Republic of Germany.
Zeppezauer
M
M
D015082:2,2'-Dipyridyl / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D001576:Benzophenoneidum / Q000378:metabolism; D001665:Binding Sites; D006736:Horses; D006860:Hydrogen Bonding; D007093:Imidazoles / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D010618:Phenanthrolines / Q000378:metabolism; D011487:Protein Conformation; D014867:Water
D007093:Imidazoles; D007527:Isoenzymes; D008024:Ligands; D010618:Phenanthrolines; D014867:Water; D001576:Benzophenoneidum; D015082:2,2'-Dipyridyl; C029899:imidazole; D000426:Alcohol Dehydrogenase; C025205:1,10-phenanthroline