NAD-phenol complex formation, the inhibition of malate dehydrogenase by phenols, and the influence of phenol substitutents on inhibitory effectiveness.

Kinetic analyses indicate that inhibition by phenols of the forward reaction of malate dehydrogenase involves the binding of two molecules of phenol. One is bound as phenol, the other as a charge transfer complex of phenol with NAD. Inhibition of the reverse reaction by phenol involves the binding of only a single phenol molecule per active unit of enzyme. Kinetic evidence for this binding pattern is supported by spectral evidence in which ultraviolet absorbance and circular dichroism studies show binding of the NAD-phenol complex by malate dehydrogenase. Circular dichroism difference spectra indicate that phenol alone also binds to malate dehydrogenase.The apparent inhibition constants for fourteen variously substituted phenols were found to be significantly correlated with the hydrophobic binding constant (π), the Hammet σ function and the NAD-phenol charge transfer association constant of the individual phenols. The degree of dependency of the apparent K_i on the hydrophobicity of phenols suggests that the observed inhibition occurs via binding of phenol and/or NAD-phenol complex in hydrophobic regions of the malate dehydrogenase molecule.