STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE METAL-BINDING SITES OF CLOSTRIDIUM-THERMOCELLUM ENDOGLUCANASE CELD

Crystallographic analysis indicated that Clostridium thermocellum endoglucanase CelD contained three Ca2+-binding sites, termed A, B, and C, and one Zn2+ binding site, The protein contributed five, six, and three of the coordinating oxygen atoms present at sites A, B, and C, respectively, Proteins altered by mutation in site A (CelD(D246A)), B (CelD(D361A)), or C (CelD(D523A)) were compared with wild type CelD, The Ca2+-binding isotherm of wad type CelD was compatible with two high affinity sites (K-alpha = 2 x 10(6) M(-1)) and one low affinity site (K alpha < 10(5) M(-1)), The Ca2+-binding isotherms of the mutated proteins showed that sites A and B were the two high affinity sites and that site C was the low affinity site, Atomic absorption spectrometry confirmed the presence of one tightly bound Zn2+ atom per CelD molecule, The inactivation rate of CelD at 75 degrees C was decreased 1.9-fold upon increasing the Ca2+ concentration from 2 x 10(-5) to 10(-3) M. The K-m of CelD was decreased 1.8-fold upon increasing the Ca2+ concentration from 5 x 10(-6) to 10(-4) M. Over similar ranges of concentration, Ca2+ did not affect the thermostability nor the kinetic properties of Cell)D-523A. These findings suggest that Ca2+ binding to site C stabilizes the active conformation of CelD in agreement with the close vicinity of site C to the catalytic center.