A single amino acid substitution in the pleckstrin homology domain of phospholipase C δ1 enhances the rate of substrate hydrolysis

The pleckstrin homology (PH) domain has been postulated to serve as an anchor for enzymes which operate at a lipid-water interface. A lysine residue was substituted for glutamic acid in the PH domain of PLC δ1 at position 54 (E54K). Purified native and mutant enzymes were characterized using a PIP₂/dodecyl maltoside mixed micelle assay and kinetics measured according to the dual phospholipid model of Hendrickson and Dennis. Our results show that both PLC δ1 and E54K bind PIP₂ cooperatively (Hill coefficient, n =2.2 and 2.0 respectively). However, E54K shows a dramatically increased rate of PIP₂-stimulated PIP₂ hydrolysis (interfacial V_max for PLC δ1=4.9 μmol/min/mg and for E54K=31 μmol/min/mg). Also, E54K has a higher affinity for micellar substrate (equilibrium dissociation constant, K₅ =85.0 μM for E54K and 211.5 μM for PLC δ1). Centrifugation binding assays using large unilamelar phospholipid vesicles confirm that E54K binds PIP₂ with higher affinity than native enzyme. E54K is more active even though the interfacial Michaelis constant (K_m) for E54K (.034 mole fraction PIP₂) is higher than the K_m for native enzyme (0.012 mole fraction PIP₂). Finally D-IP₃ is less potent at inhibiting E54K PIP₂ hydrolysis compared to native enzyme. These results demonstrate that a single amino acid substitution in the PH domain of PLC δ1 can dramatically enhance enzyme activity. Additionally, the marked increase in V_maxfor E54K argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.