We have used citrate synthase from Thermoplasma (Tp.) acidophilum as a thermostable model system to investigate the role of hydrophobic interactions in dimer interface for maintaining high temperature stability. Three mutant enzymes were constructed by single amino acid substitutions in the interface helices: Ala97 --> Ser, Ala104 --> Thr, and Gly209 --> Ala. All of the mutations enhanced the thermostability of Tp. citrate synthase, while improving its catalytic properties (K-m, V-max, and specific activity). The highest thermostability was achieved by the Gly209 --> Ala substitution. The half-life of irreversible inactivation of the G209A mutant enzyme at 85 degrees C was about 57 min, and the midpoint of guanidinium chloride (GdmCl) induced irreversible denaturation was at 2.0 M GdmCl. Our results showed that amino acid substitutions increasing or decreasing interface hydrophobicity could further increase the thermostability of the Tp. citrate synthase. Thus, interface substitutions affecting the entropy of the unfolded state did not prove to be so critical in protein thermostabilization at higher temperatures. (C) 1998 Academic Press.