REGULATORY PROPERTIES OF A PLANT NAD - ISOCITRATE DEHYDROGENASE - EFFECT OF INORGANIC IONS

Some properties of NAD: isocitrate dehydrogenase from swede (Brassica napus L.) are described. This enzyme shows simpler regulatory properties than that from animals or fungi. Isocitrate, citrate and tricarballylate are positive effectors but neither ADP nor AMP have any effect. Pyruvate, acetate, α‐ketoglutarate, succinate and dl‐malate do not stimulate or inhibit at 2 mM. ATP and other nucleoside triphosphates inhibit this enzyme but the inhibition appears to be due to the formation of a complex with the activating cation. The principal control of the enzyme is a potent inhibition by NADH competitive with NAD. Elevation of either isocitrate or activating cation concentration lowers the Km for NAD and in this respect MnSO4 is more effective than MgSO4. In contrast, the Ki for NADH is 0.05 mM with 1 mM MgSO4 and 0.16 mM with 1 mM MnSO4. The enzyme is inhibited by high concentrations (> 20 mM) of inorganic anions. This inhibition is apparently competitive with isocitrate but anions most likely cause a conformational change. The interactions between isocitrate and activating cation have been examined. Elevation of the concentration of either isocitrate or cation raises the affinity of the enzyme for the other to a limiting value. The nature of the cation has only a slight effect on the minimum Km for isocitrate (0.35 mM with MgSO4, 0.25 mM with MnSO4) but the minimum value of the Km for MgSO4 is 100 μM compared with 7 μM for MnSO4. It is concluded that the enzyme binds one molecule each of isocitrate, activating cation and NAD or NADH at the catalytic site and one molecule of citrate or isocitrate at an effector site. Copyright © 1969, Wiley Blackwell. All rights reserved