Crystal structure of the Y52F/Y73F double mutant of phospholipase A 2 : Increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines

Abstract The enzyme phospholipase A 2 (PLA2) catalyzes the hydrolysis of the sn‐2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp‐99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr‐52 and ‐73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X‐ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 å resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3 1 21, with cell parameters α = b = 46.3 å and c = 102.95 å. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R (sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R ‐factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the α‐carbon atoms between the double mutant and wild type was 0.56 å. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro‐68. However, the hydrogen bonds of the catalytic triad, His‐48, Asp‐99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe‐52 moves toward His‐48 and Asp‐99 of the catalytic diad, and Phe‐73 moves toward Met‐8, both by about 0.5 å. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.