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 Vmax 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 (Km) for E54K (.034 mole fraction PIP2) is higher than the Km 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 Vmaxfor E54K argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.
|Original language||English (US)|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology