Electrostatic effects in the kinetics of coenzyme binding to isozymes of alcohol dehydrogenase from horse liver.
The kinetic mechanism for the binding of NAD+ and NADH to the EE and SS isozymes of alcohol dehydrogenase (LADH) was studied between pH 7 and pH 10 by monitoring the quenching of tryptophan fluorescence. A consistent interpretation of all data was only possible by introducing a two-step binding mechanism. The first binding step is related to docking of the adenosine part of the coenzymes and the subsequent isomerization to the binding of the nicotinamide part. At high NADH concentrations an additional slow isomerization was identified as a conformational transition of the protein. A pH dependence for NADH binding is observed which is restricted to changes in the binding kinetics of the adenosine moiety going from pH 7 to pH 10, a tendency which is similar also for NAD+. This is attributed to pH-dependent variations in electrostatic attractions acting as a steering force of the docking process. The nicotinamide docking of NADH is equally fast for both isozymes and pH-independent over the measured range, whereas this docking equilibrium for NAD+ is pH-dependent for EE- and SS-LADH alike and the rate of association comparable. Presumably, a GluEE-366-LysSS substitution results in a stronger binding and faster association of both oxidized and reduced cofactor to the SS isozyme. A structural proof is presented for coenzyme-competitive binding of a sulfate ion, resulting in electrostatic shielding.
Adolph
H W
HW
Department of Biochemistry, Universität des Saarlandes, D-66041 Saarbrücken, FRG. hwaldolph@rz.uni-sb.de
Kiefer
M
M
Cedergren-Zeppezauer
E
E
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism*; D011487:Protein Conformation; D055672:Static Electricity
D007527:Isoenzymes; D009243:NAD; D000426:Alcohol Dehydrogenase