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Purification and Properties of the α-Ketoglutarate Dehydrogenase Complex of Cauliflower Mitochondria

1970; Elsevier BV; Volume: 245; Issue: 21 Linguagem: Inglês

10.1016/s0021-9258(18)62711-5

1083-351X

Lawrence L. Poulsen, Randolph T. Wedding,

Biochemical and Molecular Research

Abstract A procedure is described by which the α-ketoglutarate dehydrogenase-lipoyl transsuccinylase portion of the α-ketoglutarate dehydrogenase complex of cauliflower mitochondria has been purified to a specific activity g3. The enzymatic activity is totally dependent on α-ketoglutarate, DPN, reduced CoA, thiamine pyrophosphate, Mg2+ or Ca2+, and lipoyl dehydrogenase. Either cauliflower or pig heart lipoyl dehydrogenase will couple with the α-ketoglutarate dehydrogenase-lipoyl transsuccinylase complex to produce DPNH and succinyl-CoA. Because the cauliflower enzyme as prepared has a requirement for both thiamine-PPi and Mg2+, it was possible to show that the enzyme binds magnesium-thiamine-PPi rather than free Mg2+ and free thiamine-PPi. The enzyme-magnesium-thiamine-PPi complex is relatively stable, with a half-life of about 2 min. The Km for magnesium-thiamine-PPi determined on the basis of initial rates was 8.5 x 10-6 m, while the Km determined by measurement of the length of the lag between the addition of magnesium-thiamine-PPi and the initiation of the reaction was 6.1 x 10-6 m. This close agreement supports the conclusion that the lag phase found when the reaction is initiated with magnesium-thiamine-PPi is due to the slow formation of the enzyme-magnesium-thiamine-PPi complex. As usually isolated, the α-ketoglutarate dehydrogenase-lipoyl transsuccinylase complex activity is apparently limited by an inadequate level of lipoyl dehydrogenase still complexed with the other two enzymes. When the α-ketoglutarate dehydrogenase-lipoyl transsuccinylase was isolated completely free of lipoyl dehydrogenase, it was possible to saturate this complex by the addition of lipoyl dehydrogenase either from cauliflower or from pig heart. When the lipoyl dehydrogenase is present in excess it is possible accurately to determine the kinetic parameters for the substrates and cofactors of the complex.