Cold-active citrate synthase: mutagenesis of active-site residues

A comparison of the crystal structure of the dimeric enzyme citrate synthas e from the psychrophilic Arthrobacter strain DS2-3R with that of the struct urally homologous enzyme from the hyperthermophilic Pyrococcus furiosus rev eals a significant difference in the accessibility of their active sites to substrates. In this work, we investigated the possible role in cold activi ty of the greater accessibility of the Arthrobacter citrate synthase. By si te-directed mutagenesis, we replaced two alanine residues at the entrance t o the active site with an arginine and glutamate residue, respectively, as found in the equivalent positions of the Pyrococcus enzyme Also, we introdu ced a loop into the active site of the psychrophilic citrate synthase, agai n mimicking the situation in the hyperthermophilic enzyme. Analysis of the thermoactivity and thermostability of the mutant enzymes reveals that cold activity is not significantly compromised by the mutations, but rather the affinity for one of the substrates, acetyl-CoA, is dramatically increased. Moreover, one mutant (Loop insertion/K313L/A361R) has an increased thermost ability but a reduced temperature optimum for catalytic activity. This unex pected relationship between stability and activity is discussed with respec t to the nature of the dependence of catalytic activity on temperature.