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J. Biol. Chem., Vol. 278, Issue 14, 12271-12277, April 4, 2003

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Characterization and Implications of Ca²⁺ Binding to Pectate Lyase C^*

Steven R. Herron, Robert D. Scavetta^§, Michael Garrett^¶, Margaret Legner, and Frances Jurnak^**

From the Department of Physiology and Biophysics, University of California, Irvine, California 92697-4560

Ca²⁺ is essential for in vitro activity of Erwinia chrysanthemi pectate lyase C (PelC). Crystallographic analyses of 11 PelC-Ca²⁺ complexes, formed at pH 4.5, 9.5, and 11.2 under varying Ca²⁺ concentrations, have been solved and refined at a resolution of 2.2 Å. The Ca²⁺ site represents a new motif for Ca²⁺, consisting primarily of -turns and -strands. The principal differences between PelC and the PelC-Ca²⁺ structures at all pH values are the side-chain conformations of Asp-129 and Glu-166 as well as the occupancies of four water molecules. According to calculations of pK_a values, the presence of Ca²⁺ and associated structural changes lower the pK_a of Arg-218, the amino acid responsible for proton abstraction during catalysis. The Ca²⁺ affinity for PelC is weak, as the K_d was estimated to be 0.132 (±0.004) mM at pH 9.5, 1.09 (±0.29) mM at pH 11.2, and 5.84 (±0.41) mM at pH 4.5 from x-ray diffraction studies and 0.133 (±0.045) mM at pH 9.5 from intrinsic tryptophan fluorescence measurements. Given the pH dependence of Ca²⁺ affinity, PelC activity at pH 4.5 has been reexamined. At saturating Ca²⁺ concentrations, PelC activity increases 10-fold at pH 4.5 but is less than 1% of maximal activity at pH 9.5. Taken together, the studies suggest that the primary Ca²⁺ ion in PelC has multiple functions.

The atomic coordinates and the structure factors (code 1O88, 1O8D, 1O8E, 1O8F, 1O8G, 1O8H, 1O8I, 1O8J, 1O8K, 1O8L, and 1O8M) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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