Thermostability and thermoactivity of citrate synthases from the thermophilic and hyperthermophilic archaea, Thermoplasma acidophilum and Pyrococcus furiosus

Citrate synthases from Thermoplasma acidophilum (optimal growth at 55 degre esC) and Pyrococcus furiosus (100 degreesC) are homo-dimeric enzymes that s how a high degree of structural homology with each other, and thermostabili ties commensurate with the environmental temperatures in which their host c ells are found. A comparison of their atomic structures with citrate syntha ses from mesophilic and psychrophilic organisms has indicated the potential importance of inter-subunit contacts for thermostability, and here we repo rt the construction and analysis of site-directed mutants of the two citrat e synthases to investigate Be contribution of these interactions. Three set s of mutants were made: (a) chimeric mutants where the large (inter-subunit contact) and small (catalytic) domains of the T. acidophilum and P. furios us enzymes were swapped; (b) mutants of the P. furiosus citrate synthase wh ere the inter-subunit ionic network is disrupted; and (c) P. furiosus citra te synthase mutants in which the C-terminal arms that wrap around their par tner subunits have been deleted. All three sets of mutant enzymes were expr essed as recombinant proteins in Escherichia coli and were found to be cata lytically active. Kinetic parameters and the dependence of catalytic activi ty on temperature were determined, and the stability of each enzyme was ana lysed by irreversible thermal inactivation experiments. The chimeric mutant s indicate that the thermostability of the whole enzyme is largely determin ed by the origin of the large, inter-subunit domain, whereas the dependence of catalytic activity on temperature is a function of the small domain. Di sruption of the inter-subunit ionic network and prevention of the C-termina l interactions both generated enzymes that were substantially less thermost able. 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 thermoa ctivity, showing that stability is necessary for, but does not guarantee, c atalytic activity at elevated temperatures. (C) 2000 Academic Press.