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Abstract
Complexes of liver alcohol dehydrogenase with pyrazole and NAD+, NHD+, or AcPyAD+ were studied by spectral, kinetic, and equilibrium binding techniques. Difference spectra showed the same peak, 290 mµ, for ternary complex with NAD+ or NHD+ as coenzyme but with AcPyAD+ as coenzyme the peak appeared at 308 mµ. The dissociation constants of coenzyme from the ternary complex were much higher for the two analogues than for NAD+. Stopped flow studies showed that for NHD+ the higher dissociation constant was due to a decreased on velocity of the coenzyme but for AcPyAD+ it was due to a higher off velocity than NAD+. The dissociation constants of pyrazole from the ternary complexes were the same for complexes with NAD+ and NHD+ but 8-fold higher for complex containing AcPyAD+. This was due to a much slower pyrazole on velocity. The results of this study indicate that pyrazole interacts with the nicotinamide ring of NAD+ in the ternary complex. The nature of this interaction is discussed.
Highlights
The dissociation constants for NHD+and NHDH from liver alcohol dehydrogenase are considerably higher than the corresponding values for NAD+ and NADH
A comparison of the binary complex dissociation constants indicates that only the NHD+ is less tightly bound to free enzyme, while for the ternary complexes both analogues are much more loosely bound
The ki values obtained by stopped flow studies correlate well with reciproca.1 4’i values, which are the ‘(on” velocities of oxidized coenzyme determined using steady state kinetics. This correlation indicates that using a ternary complex-forming substrate competitive inhibitor to diminish the dissociation constant of the coenzyme provides a valid method for determining the rate of binding of oxidized coenzyme and analogues to enzyme
Summary
Since oxidized coenzyme is loosely bound to liver alcohol dehydrogenase and the binary complex difference spectrum peak overlaps the absorption peaks of free enzyme and free NAD+, it is not possible to study the binding rates of oxidized coenzyme and coenzyme analogues directly by stopped flow methods. Binary complex dissociation constants for oxidized coenzymes and analogues, KB, o, were determined either by the competitive titration method of Theorell and The dissociation constants of coenzyme and analogues from the ternary LADH-coenzyme-pyrazole complex, KEPY,O , were determined by spectrophotometric titrations at 300 rnp for NAD+ and
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