Identification of key amino acid residues in the assembly of enzymes into the pyruvate dehydrogenase complex of Bacillus stearothermophilus: A kinetic and thermodynamic analysis

Hyo Il Jung, Alan Cooper, Richard N. Perham

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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 (ΔH° = +2.2 kcal/mol) and a large, positive entropy change (TΔS° = +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 (ΔH° = -2.6 kcal/mol) and a less positive entropy change (TΔS° = +7.2 kcal/mol). The midpoint melting temperature (Tm) of E3-PSBD complexes was determined by differential scanning calorimetry. The R135A mutation caused a significant decrease (5 °C) in the Tm, compared with the wild-type complex. The results reveal the importance of Arg 135 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 ΔCp of subunit association illustrate the difficulties in attributing changes in heat capacity to specific classes of interactions.

Original languageEnglish
Pages (from-to)10446-10453
Number of pages8
JournalBiochemistry
Volume41
Issue number33
DOIs
Publication statusPublished - 2002 Aug 20

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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