Nucleotide Activation of Cauliflower α-Ketoglutarate Dehydrogenase

The complex of α-ketoglutarate dehydrogenase with lipoyl transsuccinylase isolated from cauliflower florets and assayed with exogenous lipoyl dehydrogenase, is markedly activated by adenosine 5'-monophosphate. This activation shows an optimum at about 1 mm AMP, with higher concentrations producing a smaller increase in rate. Kinetic analysis shows the activation to be of the coupling type in which the activator binds to the enzyme-substrate complex. The presence of AMP causes an increase in the maximal velocity of the reaction, measured with variable concentrations of α-ketoglutarate, and decreases the apparent Km for α-keto-glutarate. With the use of the parameters V''max (maximal velocity at infinite activator concentration) and K''m (K''m for α-keto-glutarate at infinite activator concentration) and the ratio V''max: K''m (designated as an activation coefficient), it has been possible to compare the activation by AMP with other nucleotides and related compounds. This comparison shows that AMP is 10 times more effective than ADP, which in turn is about twice as effective as ATP, which produces an activation coefficient about twice that of the untreated control. This method of comparison of activating effectiveness reveals that adenosine nucleotides are preferred by this enzyme, that the 2' and 3' —OH groups on the ribose of the nucleotide are involved in producing a maximal activation, and that both the purine or pyrimidine ring and the phosphate are necessary for maximal effect. Inorganic phosphate produces a significant activation. Hill plots of the rate of this reaction against α-ketoglutarate concentration have a slope of 1, which is not altered by the presence of nucleotide activators. Hill plots of rate against nucleotide concentration also give a slope of 1.