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 language | English (US) |
|---|---|
| Journal | FASEB Journal |
| Volume | 12 |
| Issue number | 8 |
| State | Published - Jan 1 1998 |
Fingerprint
All Science Journal Classification (ASJC) codes
- Biotechnology
- Biochemistry
- Molecular Biology
- Genetics
Cite this
}
Specificity requirements for the second step of the reaction catalyzed by mammalian pyruvate dehydrogenase (E1). / Gong, X.; Peng, T.; Yakhnin, Alexander Victor; Quinn, J.; Veaman, S. J.; Roche, T. E.
In: FASEB Journal, Vol. 12, No. 8, 01.01.1998.Research output: Contribution to journal › Article
TY - JOUR
T1 - Specificity requirements for the second step of the reaction catalyzed by mammalian pyruvate dehydrogenase (E1)
AU - Gong, X.
AU - Peng, T.
AU - Yakhnin, Alexander Victor
AU - Quinn, J.
AU - Veaman, S. J.
AU - Roche, T. E.
PY - 1998/1/1
Y1 - 1998/1/1
N2 - 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
AB - 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
UR - http://www.scopus.com/inward/record.url?scp=33749112263&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33749112263&partnerID=8YFLogxK
M3 - Article
AN - SCOPUS:33749112263
VL - 12
JO - FASEB Journal
JF - FASEB Journal
SN - 0892-6638
IS - 8
ER -