NADP+-linked isocitrate dehydrogenase: a study of the catalytic and regulatory properties of the enzyme.

Holland, Peggy Mary (1971) NADP+-linked isocitrate dehydrogenase: a study of the catalytic and regulatory properties of the enzyme. Masters thesis, Memorial University of Newfoundland.

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Abstract

NADP-isocitrate dehydrogenase is believed to catalyze at least 3 distinct chemical reactions, to be inhibited allosterically by folate and to be inhibited in a concerted manner by mixtures of glyoxalate and oxaloacetate. Modification of the enzyme from pig heart mitochondria by various chemical reagents and X-irradiation has been used to attempt to differentiate between the sites responsible for the five apparent enzymic functions. Consistent with the views of other workers, the present results suggest that the 3 catalytic activities involve overlapping active sites with a sulfhydryl group in the region of total overlap. Furthermore, the site or sites involved in binding the inhibitors for the concerted inhibition seem separate from the catalytic sites, suggesting that this is an allosteric inhibition. The alleged allosteric inhibition of the enzyme by folate has been examined and suggested to be a non-specific effect without regulatory significance. -- Evidence is also given suggesting that like the pig liver cytosol enzyme, the pig heart mitochondrial enzyme can catalyze the NADPH linked reduction of oxaloacetate. However, this activity is very low relative to the isocitrate dehydrogenase activity. -- Sulfhydryl or methionine modification can cause inactivation of the enzyme. However, sulfhydryl modification seemed sufficient to explain the inactivation of the enzyme upon X-irradiation. The yields (G-values) for inactivation of the enzyme and for destruction of the 4 most radiosensitive sulfhydryl groups were 0.5 and 2.0 respectively, suggesting that isocitrate dehydrogenase is quite a radiosensitive enzyme possessing some of the most radiosensitive of enzyme sulfhydryl groups. The high chemical reactivity of these groups was further implied by the high sensitivity of the enzyme to various sulfhydryl reagents. Unlike most allosteric enzymes, the regulatory properties of isocitrate dehydrogenase were not more radiosensitive than the catalytic properties. -- The results are discussed in terms of the binding site for the inhibitors. A possible regulatory role for the concerted inhibition is suggested and the significance of this in terms of mitochondrial isocitrate oxidation is discussed.

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