Modulation of 2-oxoglutarate dehydrogenase complex by inorganic phosphate,Mg2+, and other effectors

The interplay of inorganic phosphate (Pi) with other ligands such as Mg2+, ADP, ATP, and Ca2+ on the activation of 2-oxoglutarate dehydrogenase comple x (2-BGDH) in both isolated enzyme complex and mitochondrial extracts was e xamined. Pi alone activated the enzyme, following biphasic kinetics with hi gh (K-0.5 = 1.96 +/- 0.42 mM) and low (K-0.5 = 9.8 +/- 0.4 mM) affinity com ponents for Pi. The activation by Pi was highly pH-dependent; it increased when the pH raised from 7.1 to 7.6, but it was negligible at pH values belo w 7.1. Mg-Pi and Mg-ADP, but not Mg-ATP, were more potent activators of 2-O GDH than free Pi and free ADP. ATP inhibited the 2-OGDH activity by chelati ng the free Mg2+ and also as a Mg-ATP complex. With or without Mg2+, ADP, a nd Pi activated the 2-OGDH by increasing the affinity for 2-OG and the V-m of the reaction; ATP diminished the V-m, but it increased the affinity for 2-OG in the mitochondrial extract, Pi did not modify the 2-OGDH activation by Ca2+. The results above mentioned were similar for both preparations, ex cept for hyperbolic kinetics in the isolated enzyme and sigmoidal kinetics in the mitochondrial extracts when 2-oxoglutarate was varied. The data of t his study indicated that physiological concentrations of Pi may exert a sig nificant activation of 2-OGDH, which was potentiated by Mg2+ and high pH, b ut surpassed by ADP. (C) 2000 Academic Press.