A single amino acid substitution in the pleckstrin homology domain of phospholipase C delta1 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) delta1/PH domain enhancement-of-activity mutant was generated. A lysine residue was substituted for glutamic acid in the PH domain of PLC delta1 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 delta1 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 Vmax for PLC delta1 = 4.9 +/- 0.3 micromol/min/mg and for E54K = 31 +/- 3 micromol/min/mg) as well as PI hydrolysis (Vmax for PLC delta1 = 27 +/- 3.4 nmol/min/mg and for E54K = 95 +/- 12 nmol/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 micellar substrate (equilibrium dissociation constant, Ks = 85 +/- 36 microM for E54K and 210 +/- 48 microM for PLC delta1). 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 (Km) for E54K (0.034 +/- 0.01 mol fraction PI(4,5)P2) is higher than the Km 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 delta1 can dramatically enhance enzyme activity. Additionally, the marked increase in Vmax for E54K argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.
Bromann
P A
PA
Feinberg Cardiovascular Research Institute, Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Boetticher
E E
EE
Lomasney
J W
JW
D001798:Blood Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007700:Kinetics; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins; D017354:Point Mutation; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*
D001798:Blood Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010750:Phosphoproteins; C039077:platelet protein P47; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases