Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface

We have investigated factors affecting stability at the subunit–subunitinterface of the dimeric enzyme 3-isopropylmalate dehydrogenase(IPMDH) from Bacillus subtilis. Site-directed mutagenesis wasused to replace methionine 256, a key residue in the subunitinteraction, with other amino acids. Thermal stability againstirreversible inactivation of the mutated enzymes was examinedby analyzing the residual activity after heat treatment. Themutations M256V and M256A increased thermostability by 2.0 and6.0°C, respectively, whereas the mutations M256L and M256Ihad no effect. Thermostability of the M256F mutated enzyme was4.0°C lower than that of the wild-type enzyme. To our surprise,increasing the hydrophobicity of residue 256 within the hydrophobiccore of the enzyme resulted in a lower thermal stability. Themutated enzymes showed an inverse correlation between thermostabilityand the volume of the side chain at position 256. Based on theX-ray crystallographic structure of Escherichia coli IPMDH,the environment around M256 in the B.subtilis homolog is predictedto be sterically crowded. These results suggest that Met256prevents favorable packing. Introduction of a smaller aminoacid at position 256 improves the packing and stabilizes thedimeric structure of IPMDH. The van der Waals volume of theamino acid residue at the hydrophobic subunit interface is animportant factor for maintaining the stability of the subunit–subunitinterface and is not always optimized in the mesophilic IPMDHenzyme. Received September 3, 2002; revised June 13, 2003; accepted June 20, 2003.