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 that operate at a lipid/water interface. To understand further the relationship between the PH domain and enzyme activity, a phospholipase C (PLC) δ1/PH domain enhancement-of-activity mutant was generated. 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 phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)/dodecyl maltoside mixed micelle assay and kinetics measured according to the dual phospholipid model of Dennis and co-workers (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739; Carmen, G. M., Deems, R. A., and Dennis, E. A. (1995) J. Biol. Chem. 270, 18711-18714). Our results show that both PLC δ1 and E54K bind phosphatidylinositol bisphosphate cooperatively (Hill coefficients, n = 2.2 ± 0.2 and 2.0 ± 0.1, respectively). However, E54K shows a dramatically increased rate of (PI(4,5)P₂)-stimulated PI(4,5)P₂ hydrolysis (interfacial V(max) for PLC δ1 = 4.9 ± 0.3 μmol/min/mg and for E54K = 31 ± 3 μmol/min/mg) as well as PI hydrolysis (V(max) for PLC δ1 = 27 ± 3.4 μmol/min/mg and for E54K = 95 ± 12 μmol/min/mg). In the absence of PI(4,5)P₂ both native and mutant enzyme hydrolyze PI at similar rates. E54K also has a higher affinity for miceliar substrate (equilibrium dissociation constant, K(N) = 85 ± 36 μM for E54K and 210 ± 48 μM for PLC δ1). Centrifugation binding assays using large unilamelar phospholipid vesicles confirm that E54K binds PI(4,5)P₂ with higher affinity than native enzyme. E54K is more active even though the interfacial Michaelis constant (K(m)) for E54K (0.034 ± 0.01 mol fraction PI(4,5)P₂) is higher than the K(m) for native enzyme (0.012 ± 0.002 mol fraction PI(4,5)P₂). D-Inositol trisphosphate is less potent at inhibiting E54K PI(4,5)P₂ hydrolysis compared with 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(max) for E54K argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.