Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F |
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Authors: | Thunnissen MGM Marjolein; Franken Peet A; de Haas Gerard H; Drenth Jan; Kalk Kor H; Verheij Hubertus M; Dijkstra W |
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Affiliation: | Laboratory of Biophysical Chemistry and BIOSON Research Institute, University of Groningen Nijenborgh 4, 9747 AG Groningen, The Netherlands
1Department of Enzymology and Protein Engineering, State University of Utrecht, CBLE, University Center De Uithof Padualaan 8, 3584 CH Utrecht, The Netherlands |
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Abstract: | Tyr52 and Tyr73 are conserved amino acid residues throughoutall vertebrate phospholipases A2. They are part of an extendedhydrogen bonding system that links the N-terminal -NH+3 -groupto the catalytic residues His48 and Asp99. These tyrosines werereplaced by phenylalanines in a porcine pancreatic phospholipaseA2 mutant, in which residues 6266 had been deleted ( 6266PLA2).The mutations did not affect the catalytic properties of theenzyme, nor the folding kinetics. The stability against denaturatlonby guanidine hydrochloride was decreased, however. To analysehow the enzyme compensates for the loss of the tyrosine hydroxylgroup, the X-ray structures of the Y52F and AY73F mutants weredetermined. After crystallographic refinement the final crystallographicR-factors were 18.1% for the %Y52F mutant (data between 7 and2.3 Å resolution) and 19.1% for the Y73F mutant (databetween 7 and 2.4 Å resolution). No conformational changesoccurred in the mutants compared with the 6266PLA2, butan empty cavity formed at the site of the hydroxyl group ofthe former tyrosine. In both mutants the Asp99 side chain losesone of its hydrogen bonds and this might explain the observeddestabilization. |
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Keywords: | folding/ phospholipase A2/ Stability/ X-ray structure |
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