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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 Ca(2+)-binding sites, termed A, B, and C, and one Zn(2+)-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 (CelDD246A), B (CelDD361A), or C (CelDD523A) were compared with wild type CelD. The Ca(2+)-binding isotherm of wild type CelD was compatible with two high affinity sites (Ka = 2 x 10(6) M-1) and one low affinity site (Ka < 10(5) M-1). The Ca(2+)-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 Km 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 CelDD523A. 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.
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Sylvie Chauvaux, Hélène Souchon, Pedro Alzari, Patrick Chariot, Pierre Beguin. Structural and Functional Analysis of the Metal-binding Sites of Clostridium thermocellum Endoglucanase CelD. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 1995, 270 (17), pp.9757-9762. ⟨10.1074/jbc.270.17.9757⟩. ⟨pasteur-03136564⟩

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