title: The C terminus of mammalian phospholipase D is required for catalytic activity.
abstract: The activity of phospholipase D (PLD) is regulated by a variety of hormonal stimuli and provides a mechanistic pathway for response of cells to extracellular stimuli. The two identified mammalian PLD enzymes possess highly homologous C termini, which are required for catalytic activity. Mutational analysis of PLD1 and PLD2 reveals that modification of as little as the C-terminal threonine or the addition of a single alanine attenuates activity of the enzyme. Protein folding appears to be intact because mutant enzymes express to similar levels in Sf9 cells and addition of peptides representing the C-terminal amino acids, including the simple hexamer PMEVWT, restores partial activity to several of the mutants. Analysis of several mutants suggests a requirement for the hydrophobic reside at the -2-position but not an absolute requirement for the hydroxyl side chain of threonine at the C terminus. The inability of peptides amidated at their C termini to effect restoration of activity indicates the involvement of the C-terminal alpha carboxyl group in functional activity of these enzymes. The ability of peptides to restore activity to PLD enzymes mutated at the C terminus suggests a flexible interaction of this portion of the molecule with a catalytic core constructed on conserved HKD motifs. Participation of these C termini residues in either stabilization of the catalytic site or the enzymatic reaction itself remains to be determined. This requirement for the C terminus provides an excellent potential site for interaction with regulatory proteins that may either enhance or down-regulate the activity of these enzymes in vitro.
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
authors.forename: M Y
authors.initials: MY
authors.lastname: Liu
authors.forename: S
authors.initials: S
authors.lastname: Gutowski
authors.forename: P C
authors.initials: PC
authors.lastname: Sternweis
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D006801:Humans; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1