Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D

Abstract

Clostridium thermocellum endoglucanase D (EC 3.2.1.4; EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 106 M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the K(d) of the enzyme towards p-nitrophenyl-β-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75°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.