A single amino acid substitution in the pleckstrin homology domain of phospholipase C delta 1 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) delta 1/PH domain enhancement-of-activity mutant was generated. A lysine residue was substituted for glutamic acid in the PH domain of PLC delta 1 at position 54 (E54K). Purified native and mutant enzymes were characterized using a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2)/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 delta 1 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)P-2)-stimulated PI(4,5)P-2 hydrolysis (interfacial V-max for PLC delta 1 = 4.9 +/- 0.3 mu mol/min/mg and for E54K = 31 +/- 3 mu mol/min/mg) as well as PI hydrolysis (V-max for PLC delta 1 = 27 +/- 3.4 nmol/min/mg and for E54K = 95 +/- 12 nmol/min/mg). In the absence of PI(4,5)P-2 both native and mutant enzyme hydrolyze PI at similar rates. E54K also has a higher affinity for micellar substrate (equilibrium dissociation constant, K-s = 85 +/- 36 mu M for E54R and 210 +/- 48 mu M for PLC delta 1). Centrifugation binding assays using large unilamelar phospholipid vesicles confirm that E54K binds PI(4,5)P-2 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)P-2) is higher than the K-m for native enzyme (0.012 +/- 0.002 mol fraction PI(4,5)P-2). D-Inositol trisphosphate is less potent at inhibiting E54K PI(4,5)P-2 hydrolysis compared with native enzyme. These results demonstrate that a single amino acid substitution in the PH domain of PLC delta 1 can dramatically enhance enzyme activity. Additionally, the marked increase in V-max for E54R argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.