Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F

Abstract

Tyr52 and Tyr73 are conserved amino acid residues throughout all vertebrate phospholipases A₂. They are part of an extended hydrogen bonding system that links the N-terminal α-NH⁺₃ -group to the catalytic residues His48 and Asp99. These tyrosines were replaced by phenylalanines in a porcine pancreatic phospholipase A₂ mutant, in which residues 62–66 had been deleted (Δ62–66PLA₂). The mutations did not affect the catalytic properties of the enzyme, nor the folding kinetics. The stability against denaturatlon by guanidine hydrochloride was decreased, however. To analyse how the enzyme compensates for the loss of the tyrosine hydroxyl group, the X-ray structures of the ΔY52F and AY73F mutants were determined. After crystallographic refinement the final crystallographic R-factors were 18.1% for the %Y52F mutant (data between 7 and 2.3 Å resolution) and 19.1% for the ΔY73F mutant (data between 7 and 2.4 Å resolution). No conformational changes occurred in the mutants compared with the Δ62–66PLA₂, but an empty cavity formed at the site of the hydroxyl group of the former tyrosine. In both mutants the Asp99 side chain loses one of its hydrogen bonds and this might explain the observed destabilization.