Electrostatic effect upon association of reduced nicotinamide adenine dinucleotide and equine liver alcohol dehydrogenase

Abstract

The rate of association of equine liver alcohol dehydrogenase and its coenzymes exhibits a large pH dependence with slower rates at basic pH and an observed kinetic pKa value of approximately 9-9.5. This pH dependence has been explained by invoking local active site electrostatic effects which result in repulsion of the negatively charged coenzyme and the ionized hydroxyl anion form of the zinc-bound water molecule. We have examined a simpler hypothesis, namely, that the pH dependence results from the electrostatic interaction of the coenzyme and the enzyme which changes from an attractive interaction of the negatively charged coenzyme and the positively charged enzyme to a repulsive interaction between the two negatively charged species at the isoelectric point for the enzyme (pH 8.7). We have tested this proposal by examining the ionic strength dependence of the association rate constant at various pH values. These data have been interpreted by using the Wherland-Gray equation, which we have shown can be applied to the kinetics of enzyme-coenzyme association. Our results indicate that the shielding of the buffer electrolyte changes from a negative to a positive value as the charge on the protein changes at the isoelectric point. This result is exactly that which is predicted for electrostatic effects that depend on the charge of the protein molecule and is not consistent with predictions based upon the local active site effects. At low ionic strength values of 10 mM or less, approximately 75% of the observed pH dependence results from the enzyme electrostatic effects; the remaining pH dependence may result from active site effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrostatic Effects in the Kinetics of Coenzyme Binding to Isozymes of Alcohol Dehydrogenase from Horse Liver

“…In this investigation the ionic strength is about 0.1 M, throughout. That is within a range where the attraction/repulsion forces are irrelevant according to Lively et al (1987). Still, data presented here strongly indicate that electrostatic attraction/repulsion does control the coenzyme docking process.…”

“…In spite of a long lasting discussion on the issue no definite experimental proof has been presented which could unambiguously sort out which of His-51 or zinc-bound water in the active site or Lys-228 (remote from the active site) is the relevant candidate. On the contrary, Lively et al (1987) argue that none of these three electrostatic models most favored in the literature could fit their experimental data and electrostatic calculations. They conclude that the major effect of pH at low ionic strength originates from attraction/ repulsion of the negatively charged coenzyme and the protein, the protein altering its overall charge from positive to negative values over the pH scale.…”

“…In this investigation the presence of any significant concentration of anions, able to interfere with the coenzyme binding process, was strictly avoided. Due to longer dead times and lower sensitivity of the stopped-flow equipment available it was difficult earlier to maintain a sufficiently high excess of NADH in kinetic experiments to allow evaluation of data using the pseudofirst-order approach [e.g., Lively et al (1987) and Theorell et al (1970)]. …”

Unraveling and Manipulating of NADH Oxidation by Photogenerated Holes

“…This was consistent with the unchanged potential of NADH between pH 7.0-9.0, which confirmed that the adsorption of NADH during the conversion of NADH into NADH• + was the rate-limiting step in photocatalytic NADH oxidation. 39 During photocatalytic NADH oxidation, both enzymatically active NAD + and inactive ADP-ribose derivatives were generated. This would reduce the effective electron shuttle (NADH/NAD + ) between photocatalyst and enzyme.…”

Chloride binding proteins: mechanistic implications for the oxygen-evolving complex of Photosystem II

“…Chloride activation of the human enzyme is greatly reduced by an Arg ~ His mutation at position 47 in the flz subunit (Buhler andVon Wartburg 1984, Erhig et al 1988). The influence of anions on the NADH-LADH association reaction also may involve global as well as local electrostatic shielding effects (Lively et al 1987). These examples underscore the difficulty in unequivocally demonstrating C1-binding to protein-linked metal ions (to the exclusion of other potential sites), even in isolated, purified proteins.…”