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Originally published In Press as doi:10.1074/jbc.M209306200 on January 22, 2003

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. HerronDagger , 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 beta -turns and beta -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 pKa values, the presence of Ca2+ and associated structural changes lower the pKa of Arg-218, the amino acid responsible for proton abstraction during catalysis. The Ca2+ affinity for PelC is weak, as the Kd 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.

* This work was supported by United States Department of Agriculture Grant 02-03560.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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/).

Dagger Present address: W. M. Keck Center for Molecular Structure, California State University, Dept. of Chemistry and Biochemistry, Fullerton, CA 92834.

§ Present address: S. C. Johnson Research Facility, Mayo Clinic-Scottsdale, 13400 E. Shea Blvd., Scottsdale, AZ 85259.

Present address: Dept. of Physiology and Molecular Medicine, Rm. 320, Block Health Science Bldg., Medical College of Ohio, 3035 Arlington Ave., Toledo, OH 43614.

|| Present address: 224 Sixth St., Providence, RI 02906.

** To whom correspondence should be addressed: Dept. of Physiology and Biophysics, D-350 Medical Science Bldg. I, University of California, Irvine, CA 92612. Tel.: 949-824-6623; Fax: 949-824-8540; E-mail: jurnak@uci.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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