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Cation binding to a Bacillus (1,3-1,4)-beta-glucanase. Geometry, affinity and effect on protein stability
T Keitel1, M Meldgaard, U Heinemann
1Institut für Kristallographie, Freie Universität Berlin, Germany.
European Journal of Biochemistry
|May 15, 1994
Summary
Calcium ions stabilize the hybrid Bacillus (1,3-1,4)-beta-glucanase H(A16-M) structure and activity. This cation binding, distinct from the active site, enhances enzyme function at high temperatures.
Area of Science:
- Enzymology
- Structural Biology
- Biochemistry
Background:
- The hybrid Bacillus (1,3-1,4)-beta-glucanase H(A16-M) is composed of N-terminal amino acids from B. amyloliquefaciens and C-proximal amino acids from B. macerans.
- This enzyme possesses a calcium-binding site located away from the active center.
Purpose of the Study:
- To investigate the binding characteristics of calcium and sodium ions at the remote site of H(A16-M).
- To determine the effect of calcium binding on the enzyme's structural stability and thermal activity.
Main Methods:
- X-ray diffraction analysis was used to determine the structure of H(A16-M) crystals grown with and without calcium.
- Isothermal titration calorimetry (ITC) was employed to quantify the binding affinity of calcium over sodium.
- Guanidinium chloride unfolding and thermal inactivation experiments assessed protein stability.
Main Results:
- X-ray analysis revealed that the binding site is occupied by sodium ions in the absence of calcium, with sodium exhibiting fivefold coordination.
- Calcium ions are sixfold coordinated, with two water molecules in their coordination sphere.
- Calorimetric data indicated a higher affinity of H(A16-M) for calcium compared to sodium.
- Calcium binding was shown to stabilize the enzyme's native three-dimensional structure and enhance its activity at elevated temperatures.
Conclusions:
- The study elucidates the distinct coordination and binding preferences of calcium and sodium ions at a remote site on H(A16-M).
- Calcium binding plays a crucial role in stabilizing the enzyme's structure, thereby enhancing its functional activity, particularly under thermal stress.