Citrate synthases from Thermoplasma acidophilum (optimal growth at 55 degreesC) and Pyrococcus furiosus (100 degreesC) are homo-dimeric enzymes that show a high degree of structural homology with each other, and thermostabilities commensurate with the environmental temperatures in which their host cells are found. A comparison of their atomic structures with citrate synthases from mesophilic and psychrophilic organisms has indicated the potential importance of inter-subunit contacts for thermostability, and here we report the construction and analysis of site-directed mutants of the two citrate synthases to investigate Be contribution of these interactions. Three sets of mutants were made: (a) chimeric mutants where the large (inter-subunit contact) and small (catalytic) domains of the T. acidophilum and P. furiosus enzymes were swapped; (b) mutants of the P. furiosus citrate synthase where the inter-subunit ionic network is disrupted; and (c) P. furiosus citrate synthase mutants in which the C-terminal arms that wrap around their partner subunits have been deleted. All three sets of mutant enzymes were expressed as recombinant proteins in Escherichia coli and were found to be catalytically active. Kinetic parameters and the dependence of catalytic activity on temperature were determined, and the stability of each enzyme was analysed by irreversible thermal inactivation experiments. The chimeric mutants indicate that the thermostability of the whole enzyme is largely determined by the origin of the large, inter-subunit domain, whereas the dependence of catalytic activity on temperature is a function of the small domain. Disruption of the inter-subunit ionic network and prevention of the C-terminal interactions both generated enzymes that were substantially less thermostable. Taken together, these data demonstrate the crucial importance of the subunit contacts to the stability of these oligomeric enzymes. Additionally, they also provide a clear distinction between thermostability and thermoactivity, showing that stability is necessary for, but does not guarantee, catalytic activity at elevated temperatures. (C) 2000 Academic Press.