Enzymic catalysis of the keto-enol tautomerization of phenylpyruvic acids.

The present paper describes a new enzyme from animal tissues which catalyzes the keto-enol tautomeriaation of the phenylpyruvic acids. Preliminary accounts of this tautomerase have been published (1, 2). The enzyme has not yet proved to be essential in any biological reaction, but a physiological role may be expected since specific tautomers of a-keto acids are substrates or products in some reactions (3-6), and since the spontaneous tautomerization of the phenylpyruvic acids is slow. The spontaneous reaction, from freshly dissolved aqueous solutions of the crystalline acid (enol) to equilibrated solutions (largely keto), has been studied by following the increased solubility, decreased ultraviolet absorption, and increased reaction with phenylhydrazine which occur in parallel with the tautomerization to the keto form (7-10). A new assay was developed to follow the enzymic reaction which depended upon the formation of a complex between boric acid and the enol tautomer, and which could be followed spectrophotometrically. Complexes of boric acid with a-hydroxy and cY-keto acids were studied by Beeseken and Felix (ll), but the formation of an enol-borate complex was not recognized in these earlier studies. In aqueous solutions without boric acid 96 per cent of the equilibrium mixture was the keto tautomer (I). The boric acid reacted with the enol tautomer (II) to form an enol-borate (III) with its own ultraviolet absorption. The displacement of the apparent equilibrium of the tautomerization with boric acid permitted the rate of tautomerization to be followed in the keto to enol as well as in the enol to keto direction. Since boric acid also suppressed the rate of spontaneous tautomerization, the reaction in this medium tprovided a convenient and precise enzyme assay (see the accompanying reactions).