Role of Rim Tyr/Trp Residues in Interfacial Activation of Phospholipase C Enzymes

Abstract

The Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (btPI-PLC) as a model system and mammalian PI-PLCδ1 without an intact PH domain have been examined for the contribution of rim aromatic groups to protein binding to vesicles and the correlation of this with catalytic activity. In the bacterial enzyme, two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) are critical for binding to interfaces; of the many several Tyr residues mutated, replacement with alanine at several sites (close to helix B as well as the active site) weakens membrane binding. For many of these residues the loss in binding affinity approximates what is predicted for an Ala for Trp (or Tyr) substitution. If a single Tyr is modified there is little effect on the catalytic activity measured with mM substrate (although for Y88A an increase in specific activity is seen). In the mammalian enzyme, one Trp is in the hydrophobic rim ridge and could be analogous to Trp47 in bacterial PI-PLC. Replacement of the rim Trp has little effect on binding of the protein to non-substrate containing vesicles (measured by RET of the protein to labeled PE incorporated into the vesicles). However, the activity is significantly reduced. These results (and analyses of other surface variants) are discussed in terms of the multi-domain structure of the mammalian PLC contributing to binding but with X-Y domain exhibiting similar conformational changes to the bacterial enzyme.