THE TRIGONAL AND THE ORTHORHOMBIC FORMS OF THE TRIPLE MUTANT Y52,73F/D99N OF PLA2. K. Sekar, Xin Chen, M.-D. Tsai, M. Sundaralingam, Laboratory of Biological Macromolecular Structure, Departments of Chemistry and Biochemistry, The Ohio State University, 1060 Carmack Road, Columbus, OH 43210, USA
The enzyme PLA2 hydrolyses the sn-2 ester bond of phosphoglycerides in the presence of calcium ion. The catalytic triad Asp - His - water is involved in a complex hydrogen bonding network with other active site residues that lock a structural water and provide structural support. The crystal structure of the double mutant Y52,73F1 shows the structural water, but it is missing in the single mutant D99N2. In order to understand the hydrogen bonding network around the catalytic-active site residues, we have carried out the X-ray studies of the triple mutant Y52,73F/D99N. The crystals are trigonal P3121 with a = b = 47.1Å and c= 102.4Å. 1.9Å data were collected and the refinement of the structure converged to a final R-value of 18.9% for 6809 reflections. It is interesting that the structural water is missing, as in the single mutant, and therefore the triple mutant would be expected to have similar enzymatic activity. The hydrogen bonding network is different but it has many features similar to that of the single mutant. We have also obtained crystals of the orthorhombic form3 P2l2121 with a=46.7Å, b=65.5Å and c=38.0Å. Its 1.8Å structure with the missing water is similar to the trigonal form. The structures of both forms will be compared with the single and double mutants.
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Supported by NIH grant GM45947