Specificity requirements for the second step of the reaction catalyzed by mammalian pyruvate dehydrogenase (E1)

X. Gong, T. Peng, Alexander Victor Yakhnin, J. Quinn, S. J. Veaman, T. E. Roche

Research output: Contribution to journalArticle

Abstract

The limiting stop in the mammalian pyruvate dehydrogenase complex (PDC-) is (ataly/ed by El. After decarboxylation of pyruvate. El reacts the hydroxyethylidine intermediate with a lipoate to form acetyl-dihydrolipoate. Specilui'y determining siructure of the- lipoate-carrying domain is required for the sitp in this ping pong reaction. Native and mutated forms of the outer 11 1 i or inner 1,2] of the K'2 (omponent of human P DC are tested as substrates in flic K I rea< t ion. Also structures are substituted a! the lipoylation site (K46 in I.I. K I 7.'1 in l.'2) by mutation or introduction of Hpoate analogs by lipoyl iig.-isf. Kqiiiv.ilonl changes of A ai S14 and SM1 or E52 and ¥.179 in Ll and 1.2 and mutation D172Q greatiy redncoti El catalysis. Whereas with D197A, A17-1S. next to lipoylation Mte. and D2i;îN, D219N. and E224Q. in the unioided minal region, had small effects. Removal of the terminal region xsmlially f'lhninated the El reaction. Alteration E170Q prevented lipoylation of 1.2. Lipoylation site mutants K 'o A. M or Q in 1.2 created weak competitive inhibitors V<M>IIS the native L2 with Kl7,'iQ and K173A giving 25% and 20% inhibition for 1:1 mixtures of 1,2 and mutated 1,2 at 46 //M. Similar competition if ].2 with modified L2 with lipoate replaced with o<tanoate. 8-SH-octanoate. hepianoate gave .small inhibitory effects with S-SH-octanoate being most effective (24'/r inhibition). Thus, we have detected key specificity residues in the lipoyl domain-' and obtained evidence that nopale's diihiolane ring is also a requipment.

Original languageEnglish (US)
JournalFASEB Journal
Volume12
Issue number8
StatePublished - Jan 1 1998

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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    Gong, X., Peng, T., Yakhnin, A. V., Quinn, J., Veaman, S. J., & Roche, T. E. (1998). Specificity requirements for the second step of the reaction catalyzed by mammalian pyruvate dehydrogenase (E1). FASEB Journal, 12(8).