Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex.
Unlike pyruvate dehydrogenase complexes (PDCs) from prokaryotes, PDCs from higher eukaryotes have an additional structural component, E3-binding protein (BP), for binding of dihydrolipoamide dehydrogenase (E3) in the complex. Based on the 3D structure of the subcomplex of human (h) E3 with the di-domain (L3S1) of hBP, the amino acid residues (H348, D413, Y438, and R447) of hE3 for binding to hBP were substituted singly by alanine or other residues. These substitutions did not have large effects on hE3 activity when measured in its free form. However, when these hE3 mutants were reconstituted in the complex, the PDC activity was significantly reduced to 9% for Y438A, 20% for Y438H, and 18% for D413A. The binding of hE3 mutants with L3S1 determined by isothermal titration calorimetry revealed that the binding affinities of the Y438A, Y438H, and D413A mutants to L3S1 were severely reduced (1019-, 607-, and 402-fold, respectively). Unlike wild-type hE3 the binding of the Y438A mutant to L3S1 was accompanied by an unfavorable enthalpy change and a large positive entropy change. These results indicate that hE3-Y438 and hE3-D413 play important roles in binding of hE3 to hBP.
Park
Yun-Hee
YH
Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14214, USA.
Patel
Mulchand S
MS
D002151:Calorimetry; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D019277:Entropy; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000235:genetics
C045867:PDHX protein, human; D011768:Pyruvate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase
Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1761-6
9050852
Methods Mol Biol. 1998;108:337-46
9921542
FASEB J. 1990 Nov;4(14):3224-33
2227213
J Biol Chem. 1997 Aug 8;272(32):19746-51
9242632
Biochem Soc Trans. 2003 Dec;31(Pt 6):1143-51
14641014
Biochem Soc Trans. 2006 Nov;34(Pt 5):815-8
17052205
Science. 1995 Jan 20;267(5196):383-6
7529940
J Biol Chem. 2004 Feb 20;279(8):6921-33
14638692
J Mol Biol. 1995 Apr 28;248(2):478-86
7739054
J Biol Chem. 2001 Oct 5;276(40):37223-9
11486000
J Biol Chem. 2006 Jan 6;281(1):648-55
16263718
J Mol Catal B Enzym. 2009 Nov 1;61(1-2):2-6
20160912
Structure. 2006 Mar;14(3):611-21
16442803