Evidence for Tighter Binding of Magnesium-Thiamine Pyrophosphate to α-Ketoglutarate Dehydrogenase When Activated by Adenosine Monophosphate

Abstract

Abstract The complex of α-ketoglutarate dehydrogenase with lipoyl transsuccinylase isolated from cauliflower florets and assayed with exogenous lipoyl dehydrogenase is strongly activated by adenosine 5'-monophosphate. The fact that cauliflower α-ketoglutarate dehydrogenase is free of bound thiamine pyrophosphate as isolated makes possible measurement of the influence of AMP on the binding of this cofactor to the enzyme. The presence of AMP causes an increase in the Vmax of the reaction and a decrease in the Km for Mg-thiamine-PPi. The kinetic indication of tighter binding of Mgthiamine-PPi implicit in the smaller Km is confirmed by independent and more direct measurements of the dissociation of the enzyme-cofactor complex. The half-time for dissociation of the complex of the enzyme with Mg-thiaminePPi is 5 ½ min. In the presence of AMP, the half-time of dissociation is increased to 179 min. The slower rate constant for the dissociation of the enzyme-Mg-thiamine-PPi complex is paralleled by a more rapid rate constant for the association of these components in the presence of AMP. The α-ketoglutarate dehydrogenase reaction can utilize Ca2+, Mg2+, or Sr2+, although at significantly different rates. In the presence of AMP, the rate with any of these ions is stimulated and reaches approximately the same value regardless of which ion is supplied.