PHOSPHOLIPASE-A2 ENGINEERING .10. THE ASPARTATE...HISTIDINE CATALYTIC DIAD ALSO PLAYS AN IMPORTANT STRUCTURAL ROLE

The ''catalytic triad'' of serine proteases and lipases and the ''catalytic diad'' of phospholipase A2 (PLA2) are the key catalytic machineries of these enzymes. We present evidence that the catalytic diad of PLA2 also plays an important structural role. The His-48 of PLA2 was changed to Asn, Gln, and Ala; the resulting mutants are named H48N, H48Q, and H48A, respectively. The amide nitrogen atoms of Asn and Gln mimic the delta1-nitrogen and the epsilon2-nitrogen, respectively, of His. According to the crystal structure, the delta1-nitrogen is the actual general base while the epsilon2-nitrogen is hydrogen-bonded to Asp-99. Kinetic analysis indicated that H48N retains 6 x 10(-5) of original activity while H48Q and H48A show no detectable activity. On the other hand, proton NMR and conformational stability analyses indicated that the conformation of the enzyme is largely retained in H48Q but is highly perturbed in H48N and H48A. The results suggest that while the delta1-nitrogen of His-48 is important for the catalysis by PLA2, the epsilon2-nitrogen of the same residue plays an important structural role.