Structural double-mutant cycle analysis of a hydrogen bond network in ketosteroid isomerase from Pseudomonas putida biotype B

Do Soo Jang, Hyung Jin Cha, Sun Shin Cha, Bee Hak Hong, Nam Chul Hat, Ja Young Lee, Byung Ha Oh, Heung Soo Lee, Kwan Yong Choi

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13 Scopus citations


KSI (ketosteroid isomerase) catalyses an allylic isomerization reaction at a diffusion-controlled rate. A hydrogen bond network, Asp99⋯ Waters504⋯Tyr14⋯Tyr55⋯ Tyr30, connects two critical catalytic residues, Tyr14 and Asp99, with Tyr30, Tyr55 and a water molecule in the highly apolar active site of the Pseudomonas putida KSI. In order to characterize the interactions among these amino acids in the hydrogen bond network of KSI, double-mutant cycle analysis was performed, and the crystal structure of each mutant protein within the cycle was determined respectively to interpret the coupling energy. The ΔΔGo values of Y14F/D99L (Tyr14 → Phe/Asp99 → Leu) KSI, 25.5 kJ/mol for catalysis and 28.9 kJ/mol for stability, were smaller than the sums (i.e. 29.7 kJ/mol for catalysis and 34.3 kJ/mol for stability) for single mutant KSIs respectively, indicating that the effect of the Y14F/D99L mutation was partially additive for both catalysis and stability. The partially additive effect of the Y14F/D99L mutation suggests that Tyr14 and Asp 99 should interact positively for the stabilization of the transition state during the catalysis. The crystal structure of Y14F/D99L KSI indicated that the Y14F/D99L mutation increased the hydrophobic interaction while disrupting the hydrogen bond network. The ΔΔGo. values of both Y30F/D99L and Y55F/D99L KSIs for the catalysis and stability were larger than the sum of single mutants, suggesting that either Tyr30 and Asp99 or Tyr55 and Asp99 should interact negatively for the catalysis and stability. These synergistic effects of both Y30F/D99L and Y55F/D99L mutations resulted from the disruption of the hydrogen bond network. The synergistic effect of the Y55F/D99L mutation was larger than that of the Y30F/D99L mutation, since the former mutation impaired the proper positioning of a critical catalytic residue, Tyr14, involved in the catalysis of KSI. The present study can provide insight into interpreting the coupling energy measured by double-mutant cycle analysis based on the crystal structures of the wild-type and mutant proteins.

Original languageEnglish
Pages (from-to)967-973
Number of pages7
JournalBiochemical Journal
Issue number3
StatePublished - 15 Sep 2004


  • Coupling energy
  • Double-mutant cycle
  • Hydrogen bond network
  • Ketosteroid isomerase
  • Site-directed mutagenesis


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