Studies on the enolization of pyruvate by pyruvate kinase.

Unlike the usual “kinase” reaction between ATP and an alcohol, pyruvate kinase catalyzes phosphate transfer to an oxygen which in the substrate, pyruvate, is in carbonyl linkage. It is of particular interest to ask whether enolization of pyruvate occurs sufficiently rapidly under physiological conditions so that the true substrate for the enzyme may be the enol form of pyruvate. In the work reported below it is shown that this rate is far too slow to account for the formation of phosphoenolpyruvate. If the enzyme must catalyze the enolization of pyruvate, how is this process related to the phosphorylation reaction? One might imagine that it is completely dependent on it, much as the decarboxylation of malate by malic enzyme is dependent on reduction of TPN. Enzyme reactions in which an enolization step might be expected to precede a second reaction have been found to be dependent on the presence of the second substrate, as in the condensing enzyme reaction (1,2). On the other hand, the reactions catalyzed by aldolase (3) and TPN-specific isocitric dehydrogenase (4) are known to occur by a preliminary activation of hydrogen CY to a carbonyl group in the absence of the reaction partner, n-glyceraldehyde a-phosphate and COZ, respectively. For the phosphorylation of pyruvate, ATP, Mg++, and K+ are required. What is the effect of these components on the enolization reaction, and how does the enolization rate compare with the net phosphorylation? To answer these questions a study of the activation of the methyl hydrogens of pyruvate was performed by measuring the rate of detritiation of j3-T pyruvate. The results show that enolization (hereafter, “enolization” and detritiation will be assumed to be synonymous) can, under certain conditions, be promoted by the enzyme in the absence of phosphorylation, but that all of the components necessary for the over-all reaction are also required for enolization. A number of anions are shown to replace ATP in the exchange reaction and the specificity requirements and mode of actions of these are considered. Finally, kinetic data are given for substrate specificity and for the interaction of pyruvate and ATP with the enzyme. These data, although referable to a different reaction (detritiation) are difficult to obtain by studies of the net phosphorylation of the ar-keto acid because of the rather unfavorable equilibrium of that reaction. A preliminary account of this work has appeared (5).