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Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.

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Author

Summary, in English

Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.

Publishing year

2002

Language

English

Pages

5720-5729

Publication/Series

Biochemistry

Volume

41

Issue

18

Document type

Journal article

Publisher

The American Chemical Society (ACS)

Topic

  • Biochemistry and Molecular Biology

Keywords

  • Rhodobacter : genetics
  • Rhodobacter : enzymology
  • Protein Denaturation
  • Protein Conformation
  • Protein Binding
  • Biomolecular
  • Nuclear Magnetic Resonance
  • Site-Directed
  • Mutagenesis
  • Molecular
  • Models
  • Hydrogen-Ion Concentration
  • Enzyme Stability
  • Cloning
  • Carbohydrates : metabolism
  • Calorimetry
  • Calcium : metabolism
  • Binding Sites
  • Xylosidases : chemistry
  • Xylosidases : genetics
  • Xylosidases : metabolism
  • Structure-Activity Relationship
  • Support
  • Non-U.S. Gov't
  • Temperature
  • Thermodynamics

Status

Published

ISBN/ISSN/Other

  • ISSN: 0006-2960