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Adenylatekinases （AKs） have evolved AMP-binding and lid domainsthat are encoded as continuous polypeptides embedded at differentlocations within the discontinuous polypeptide encoding the core domain.A prior study showed that AK homologues of different stabilities consistentlyretain cellular activity following circular permutation that splitsa region with high energetic frustration within the AMP-binding domaininto discontinuous fragments. Herein, we show that mesophilic andthermophilic AKs having this topological restructuring retain activityand substrate-binding characteristics of the parental AK. While permutationdecreased the activity of both AK homologues at physiological temperatures,the catalytic activity of the thermophilic AK increased upon permutationwhen assayed >30 °C below the melting temperature of the nativeAK. The thermostabilities of the permuted AKs were uniformly lowerthan those of native AKs, and they exhibited multiphasic unfoldingtransitions, unlike the native AKs, which presented cooperative thermalunfolding. In addition, proteolytic digestion revealed that permutationdestabilized each AK in differing manners, and mass spectrometry suggestedthat the new termini within the AMP-binding domain were responsiblefor the increased proteolysis sensitivity. These findings illustratehow changes in contact order can be used to tune enzyme activity andalter folding dynamics in multidomain enzymes.
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