Clostridium thermocellum endoglucanase D (EC 126.96.36.199: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3′ sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region.
Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D
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S Chauvaux, P Beguin, J P Aubert, K M Bhat, L A Gow, T M Wood, A Bairoch; Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D. Biochem J 1 January 1990; 265 (1): 261–265. doi: https://doi.org/10.1042/bj2650261
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