Coenzyme B 12-dependent propanediol dehydratase system Ternary complex between apoenzyme, coenzyme, and substrate analog

Abstract

A dead-end ternary complex was formed between propanediol dehydratase ( dl-1,2-propanediol hydro-lyase, EC 4.2.1.28) apoenzyme, coenzyme B 12, and a substrate analog when a substrate analog such as 1,2-butanediol or styrene glycol was incubated with the apoenzyme and coenzyme B 12 in the presence of potassium ions. The 1,2-diols used did not show any substrate activity, and behaved as weak competitive inhibitors with respect to the substrate. When the true substrate, 1,2-propanediol, was added in excessive amounts to the ternary complex system, the initially bound substrate analog was readily displaced by the substrate and the propanediol dehydratase reaction normally took place. The analog ternary complex was relatively stable to oxygen compared with the holoenzyme, and was much more thermostable than the apoenzyme. Like the holoenzyme and reacting holoenzyme, the ternary complex was photostable under the conditions where free coenzyme B 12 was rapidly photolyzed. The apoenzyme was completely inactivated by incubation with p- chloromercuribenzoate , iodoacetamide, or N- ethylmaleimide , but not with arsenite, suggesting that sulfhydryl groups, but not vicinal ones, are involved at the active site of the enzyme. Treatment with mercaptoethanol or dithiothreitol reversed the inhibition by p- chloromercuribenzoate . In contrast to the mercurial-insensitivity of both the holoenzyme and reacting holoenzyme, the analog ternary complex was considerably sensitive to p- chloromercuribenzoate . Upon Sephadex G-25 gel filtration, dissociation of coenzyme B 12 from the ternary complex occurred more readily than that from the holoenzyme. These results suggest that the apoenzyme-coenzyme-substrate analog ternary complex is a suitable, stable model for the so-called Michaelis complex (intermediate enzyme-substrate complex), and that its structure may be somewhat distorted compared to the holoenzyme and reacting holoenzyme.