Interaction between Pyridoxamine 5′-Phosphate and Apo-Aspartate Aminotransferase from Pig Heart

Evidence for a Negative Cooperativity

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Abstract

Apoenzyme of aspartate aminotransferase prepared according to various techniques can bind the pyridoxamine-P coenzyme and recover its full activity. An anticooperativity is observed between the two sites of the dimer; the intrinsic dissociation constants are 20 nM and 200 nM for apoenzymes prepared in the presence of phosphate and less than 1.8 and 6 nM for an apoenzyme prepared without phosphate. The phosphate ions can bind to two sites of the apoenzyme with an intrinsic constant of 1.6 μM; a competition is observed between pyridoxamine-P and phosphate but it is not sufficient to explain the observed differences between the various apoenzymes. At pH 8.3, pyridoxamine-P can dissociate from the holoenzyme: the dissociation rate is very slow so that it does not occur in enzymatic transamination.

In the presence of succinate, the two sites become equivalent and the affinity for pyridoxamine-P is smaller, meaning the occurence of an interaction between the substrate-binding site and the coenzyme-binding site.

Ancillary