Effect of pH on Pyrazole Binding to Liver Alcohol Dehydrogenase

Abstract

1. Kinetic and equilibrium data have been determined at different pH between 4 and 10 for binding of the inhibitor pyrazole to liver alcohol dehydrogenase and to the binary complexes formed between enzyme and NADH or NAD '. 2. Pyrazole binding to free enzyme requires the protonated form of an ionizing group with a pK, of 9.2, agreeing with the pKa value reported for the water molecule bound at the catalytic zinc ion of the enzyme subunit. The rate of association of the inhibitor to the enzyme . NAD' complex exhibits a similar pKa-7.6-dependence attributable to ionization of zinc-bound water in the latter binary complex. These observations lend support to the idea that pyrazole combines to the catalytic zinc ion on complex formation with the enzyme, zinc-bound water most likely being displaced by the inhibitor.3. The rate of dissociation of the inhibitor from the ternary enzyme . NAD' . pyrazole complex is proportional to the hydrogen ion concentration over the examined pH range (4-8). This effect of pH, which is proposed to reflect ionization of the enzyme-bound inhibitor with a pK, value below 4 (indirectly estimated to 2.4), accounts for the exceptional stability of the ternary complex at neutral and alkaline pH. It is concluded that pyrazole, by analogy to water and alcohol ligands, undergoes a drastic pK, perturbation on binding to the catalytic zinc ion in the enzyme . NAD' complex.Pyrazole is an effective inhibitor of the liver alcohol dehydrogenase reaction, strictly competitive with the alcohol substrate [l]. Theorell et al. demonstrated that pyrazole inhibition is attributable to the formation of an exceptionally firm enzyme . NAD' . pyrazole complex showing a characteristic difference absorption band at about 295 nm [2]. Due to the stability and chromophoric properties of this ternary complex, pyrazole has turned out to be a most useful ligand from a methodological point of view. Transient-state kinetic methods based on the absorbance changes associated with pyrazole binding have been applied to monitor formation of the enzyme . NAD' complex during catalysis and to study the process of ternary-complex formation with non-chromophoric substrate and coenzyme analogues [3-51. The tight binding of pyrazole in the ternary complex can be utilized to render NAD' dissociation insignificant in active-site titrations of the enzyme [2] and in kinetic studies of coenzyme binding [6]. The high affinity of the enzyme . NAD' complex for pyrazole makes it possible also to examine the kinetics of enzymic aldehyde reduction under single-turnover conditions by the 'catalytic suicide' method of McFarland and Bernhard [7].Despite the extensive use of pyrazole for such methodological purposes in experiments carried out at different pH, detailed kinetic information about pyrazole binding to the enzyme . NAD' complex is available only for the reaction at pH 7 [ 3 ] . The present investigation was undertaken to characterize the kinetics of the binding process over the wide pH range (4-10) usually considered in mechanistic stu...

Electrostatic field effects of coenzymes on ligand binding to catalytic zinc in liver alcohol dehydrogenase

“…A Durrum D-llO stopped-flow spectrometer, equipped as detailed previously [14], was used for the study of rapid reactions. Other nieasurements were made with a Shimadzu UV-240 spectrophotometer.…”

“…Complex formation between enzyme and the latter carboxylates was found to result in a 40-fold stabilization of N A D + binding and a slightly larger destabilization of the binding of NADH [6]. If these effects can be assumed to derive from charge interactions between coenzyme and a zinc-bound carboxylate anion, then it would seem sufficiently well established that (and how) coenzyme binding is controlled by the ionization state of zinc-bound Enzyme.…”

“…Evidence suggesting that zinc-bound anions may give rise to electrostatic field effects of that order of magnitude has been obtained by examination of' the synergism between coenzyme binding and the binding of negatively charged substrate analogues such as decanoate and benzoate [5,6]. Complex formation between enzyme and the latter carboxylates was found to result in a 40-fold stabilization of N A D + binding and a slightly larger destabilization of the binding of NADH [6].…”

“…We have attributed the observed pH dependence of coenzyme binding to electrostatic interactions reflecting ionization of the water molecule which is bound at the catalytic zinc ion of the enzyme subunit [3]. Charged groups on NADH and NAD' were assumed to interact strongly enough with a zinc-bound hydroxyl ion to account for the pK, values assigned to zincbound water in different enzyme species [4]. This would require that ionization of zinc-bound water increases (decreases) the affinity of the enzyme for NAD' (NADH) by a factor of 40 (100).…”

“…Charged groups on NADH and NAD' were assumed to interact strongly enough with a zinc-bound hydroxyl ion to account for the pK, values assigned to zincbound water in different enzyme species [4]. This would require that ionization of zinc-bound water increases (decreases) the affinity of the enzyme for NAD' (NADH) by a factor of 40 (100).Evidence suggesting that zinc-bound anions may give rise to electrostatic field effects of that order of magnitude has been obtained by examination of' the synergism between coenzyme binding and the binding of negatively charged substrate analogues such as decanoate and benzoate [5,6]. Complex formation between enzyme and the latter carboxylates was found to result in a 40-fold stabilization of N A D + binding and a slightly larger destabilization of the binding of NADH [6].…”