Abstract
Thermal unfolding kinetics of β-glucosidase B from Paenibacillus polymyxa and its thermoresistant mutant H62R were determined from far-UV circular dichroism (CD) measurements at different temperatures. The unfolding of both enzymes followed simple two-state kinetics. The new ionic pair formed between Arg62 and Glu429 in the H62R variant did not change substantially the enzyme structure as judged by far-UV CD and fluorescence spectra, but produced an increase in the unfolding activation barrier of 0.95 ± 0.10 kcal mol-1, in good agreement with the energetic contribution reported for surface salt bridges in proteins. Eyrings analysis of the unfolding kinetic constants showed that the activation enthalpies for thermal denaturation of both enzymes were essentially the same. Thus, the greater kinetic stability rendered by the salt bridge seems to be due to A reduction in the activation entropy.
Keywords: Activation entropy, hydration entropy, salt bridge, thermal unfolding kinetics, transition state
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