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

Paul A. Bromann, Evan E. Boetticher, Jon W. Lomasney*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)16240-16246
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number26
DOIs
StatePublished - Jun 27 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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