Structural and functional analysis of the metal-binding sites of Clostridium thermocellum endoglucanase CelD

Abstract

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 wild type CelD was compatible with two high affinity sites (K(a) = 2 x 106 M-1) and one low affinity site (K(a) < 105 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 °C was decreased 1.9-fold upon increasing the Ca2+ concentration from 2 x 10-5 to 10-8 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 CelD(D523A). 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.