Mutating His²⁹, His¹²⁵, His¹³³ or His¹⁵⁸ abolishes glycosylphosphatidylinositol-specific phospholipase D catalytic activity

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) specifically cleaves GPIs. This phospholipase D is a secreted protein consisting of two domains: an N-terminal catalytic domain and a predicted C-terminal β-propeller. Although the biochemical properties of GPI-PLD have been extensively studied, its catalytic site has not been identified. We hypothesized that a histidine residue(s) may play a critical role in the catalytic activity of GPI-PLD, based on the observations that (i) Zn²⁺, which utilizes histidine residues for binding, is required for GPI-PLD catalytic activity, (ii) a phosphohistidine intermediate is involved in phospholipase D hydrolysis of phosphatidylcholine, (iii) computer modelling suggests a catalytic site containing histidine residues, and (iv) our observation that diethyl pyrocarbonate, which modifies histidine residues, inhibits GPI-PLD catalytic activity. Individual mutation of the ten histidine residues to asparagine in the catalytic domain of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His⁵⁶ or His⁸⁸), secreted with catalytic activity (His³⁴, His⁸¹, His⁹⁸ or His²¹⁹) and secreted without catalytic activity (His²⁹, His¹²⁵, His¹³³ or His¹⁵⁸). Changing His¹³³ but not His²⁹, His¹²⁵ or His¹⁵⁸ to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His¹³³ is involved in Zn²⁺ binding. His¹³³ and His¹⁵⁸ also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His²⁹, His¹²⁵, His¹³³ and His¹⁵⁸ are required for GPI-PLD catalytic activity.