title: 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.
authors.affiliation: URA 1300 CNRS, Département des Biotechnologies, Institut Pasteur, Paris, France.
authors.forename: S
authors.initials: S
authors.lastname: Chauvaux
authors.forename: H
authors.initials: H
authors.lastname: Souchon
authors.forename: P M
authors.initials: PM
authors.lastname: Alzari
authors.forename: P
authors.initials: P
authors.lastname: Chariot
authors.forename: P
authors.initials: P
authors.lastname: Beguin
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D007700:Kinetics; D011487:Protein Conformation; D013696:Temperature; D015032:Zinc / Q000378:metabolism*
chemical_list: C087879:endoglucanase CelD; D002480:Cellulase; D015032:Zinc; D002118:Calcium