Design, X-ray crystallography, molecular modelling and thermal stability studies of mutant enzymes at site 172 of 3-isopropylmalate dehydrogenase from Thermus thermophilus

The relationship between the structure and the thermostability of the 3-iso propylmalate dehydrogenase from Thermus thermophilus was studied by site-di rected mutation of a single Ala residue located at the domain interface. Th e crystal structures of three mutant enzymes, replacing Ala172 with Gly, Va l and Phe, were successfully determined at 2.3, 2.2 and 2.5 Angstrom resolu tion, respectively. Substitution of Ala172 by relatively 'short' residues ( Gly, Val or Ile) enlarges or narrows the cavity in the vicinity of the C-be ta atom of Ala172 and the thermostability of the enzyme shows a good correl ation with the hydrophobicity of the substituted residues. Substitution of Ala172 by the 'longer' residues Leu or Phe causes a rearrangement of the do main structure, which leads to a higher thermostability of the enzymes than that expected from the hydrophobicity of the substituted residues. Mutatio n of Ala172 to negatively charged residues gave an unexpected result: the m elting temperature of the Asp mutant enzyme was reduced by 2.7 K while that of the Glu mutant increased by 1.8 K. Molecular-modelling studies indicate d that the glutamate side chain was sufficiently long that it did not act a s a buried charge as did the aspartate, but instead protruded to the outsid e of the hydrophobic cavity and contributed to the stability of the enzyme by enhancing the packing of the local side chains and forming an extra salt bridge with the side chain of Lys175.