title: Identification of key amino acid residues in the assembly of enzymes into the pyruvate dehydrogenase complex of Bacillus stearothermophilus: a kinetic and thermodynamic analysis.
abstract: Structural studies have shown that electrostatic interactions play a major part in the binding of dihydrolipoyl dehydrogenase (E3) to the peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acyltransferase (E2) in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The binding is characterized by a small, unfavorable enthalpy change (deltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TdeltaS degrees = +14.8 kcal/mol). The contributions of individual surface residues of the PSBD of E2 to its interaction with E3 have been assessed by alanine-scanning mutagenesis, surface plasmon resonance detection, and isothermal titration calorimetry. The mutation R135A in the PSBD gave rise to a significant decrease (120-fold) in the binding affinity; two other mutations (R139A and R156A) were associated with smaller effects. The binding of the R135A mutant to E3 was accompanied by a favorable enthalpy (deltaH degrees = -2.6 kcal/mol) and a less positive entropy change (TdeltaS degrees = +7.2 kcal/mol). The midpoint melting temperature (T(m)) of E3-PSBD complexes was determined by differential scanning calorimetry. The R135A mutation caused a significant decrease (5 degrees C) in the T(m), compared with the wild-type complex. The results reveal the importance of Arg135 of the PSBD as a key residue in the molecular recognition of E3 by E2, and as a major participant in the overall entropy-driven binding process. Further, the effects of mutagenesis on the deltaCp of subunit association illustrate the difficulties in attributing changes in heat capacity to specific classes of interactions.
authors.affiliation: Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.
authors.forename: Hyo-Il
authors.initials: HI
authors.lastname: Jung
authors.forename: Alan
authors.initials: A
authors.lastname: Cooper
authors.forename: Richard N
authors.initials: RN
authors.lastname: Perham
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics; D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D002151:Calorimetry; D002942:Circular Dichroism; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004795:Enzyme Stability / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000235:genetics; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry
chemical_list: D000596:Amino Acids; D011768:Pyruvate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D013917:Thrombin; D000409:Alanine