tailieunhanh - Báo cáo khoa học: Interactions of the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The enzymes pyruvate decarboxylase (E1) and dihydro-lipoyl dehydrogenase (E3) bind tightly but in a mutually exclusive manner to the peripheral subunit-binding domain (PSBD) of dihydrolipoyl acetyltransferase in the pyruvate dehydrogenase multienzyme complex of Bacillus stearo-thermophilus. The use of directed mutagenesis, surface plasmon resonance detection and isothermal titration microcalorimetry revealed that several positively charged residues of the PSBD, most notably Arg135, play an important part in the interaction with both E1 and E3, whereas Met131 makes a significant contribution to the binding of E1 only. . | Eur. J. Biochem. 270 4488-4496 2003 FEBS 2003 doi Interactions of the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus Hyo-Il Jung1 Alan Cooper2 and Richard N. Perham1 1 Cambridge Centre for Molecular Recognition Department of Biochemistry University of Cambridge UK 2Department of Chemistry University of Glasgow UK The enzymes pyruvate decarboxylase E1 and dihydro-lipoyl dehydrogenase E3 bind tightly but in a mutually exclusive manner to the peripheral subunit-binding domain PSBD of dihydrolipoyl acetyltransferase in the pyruvate dehydrogenase multienzyme complex of Bacillus stearo-thermophilus. The use of directed mutagenesis surface plasmon resonance detection and isothermal titration microcalorimetry revealed that several positively charged residues of the PSBD most notably Arg135 play an important part in the interaction with both E1 and E3 whereas Met131 makes a significant contribution to the binding of E1 only. This indicates that the binding sites for E1 and E3 on the PSBD are overlapping but probably significantly different and that additional hydrophobic interactions may be involved in binding E1 compared with E3. Arg135 of the PSBD was also replaced with cysteine R135C which was then modified chemically by alkylation with increasingly large aliphatic groups R135C -methyl -ethyl -propyl and -butyl . The pattern of changes in the values of AG AH and TAS that were found to accompany the interaction with the variant PSBDs differed between E1 and E3 despite the similarities in the free energies of their binding to the wild-type. The importance of a positive charge on the side-chain at position 135 for the interaction of the PSBD with E3 and E1 was apparent although lysine was found to be an imperfect substitute for arginine. The results offer further evidence of entropyenthalpy compensation .