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• W2003246046 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 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,K s = 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. 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 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,K s = 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. In many cell types, ligand binding to integral membrane receptors leads to an increase in the intracellular second messengers, inositol 1,4,5-trisphosphate (IP3) 1The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PKC protein kinase C; PH domain, pleckstrin homology domain; PI, phosphatidylinositol; PE, phosphatidylethanolamine; LUV, large unilamellar vesicle; d-IP3,d-myoinositol 1,4,5-trisphosphate; PC, phosphatidylcholine; PS, phosphatidylserine. 1The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PKC protein kinase C; PH domain, pleckstrin homology domain; PI, phosphatidylinositol; PE, phosphatidylethanolamine; LUV, large unilamellar vesicle; d-IP3,d-myoinositol 1,4,5-trisphosphate; PC, phosphatidylcholine; PS, phosphatidylserine. and diacylglycerol. This increase results largely from activation of a family of phosphoinositide-specific phospholipase C (PLC) enzymes that hydrolyze polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (3Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3307) Google Scholar). IP3 releases intracellular Ca2+ from the endoplasmic reticulum via interaction with a specific receptor located on the surface of the endoplasmic reticulum (3Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3307) Google Scholar, 4Berridge M.J. Irvine R.F. Nature. 1984; 312: 315-321Crossref PubMed Scopus (4238) Google Scholar). Diacylglycerol, as well as increased intracellular Ca2+, activates protein kinase C (PKC) (5Yagisawa H. Tanase H. Nojima H. J. Hypertens. 1991; 9: 997-1004Crossref PubMed Scopus (31) Google Scholar). Recent studies suggest that PLC δ1 can be stimulated both by a p122-Rho-GTPase-activating protein (6Homma Y. Emori Y. EMBO J. 1995; 14: 286-291Crossref PubMed Scopus (189) Google Scholar) and also by Gh(transglutaminase II) (7Feng J.-F. Rhee S.G. Im M.-J. J. Biol. Chem. 1996; 271: 16451-16454Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Recent genetic studies in spontaneously hypertensive rats suggest that activity of this effector enzyme plays an important role in the control of blood pressure (5Yagisawa H. Tanase H. Nojima H. J. Hypertens. 1991; 9: 997-1004Crossref PubMed Scopus (31) Google Scholar, 8Kato H. Fukami K. Shibasaki F. Homma Y. Takenawa T. J. Biol. Chem. 1992; 267: 6483-6487Abstract Full Text PDF PubMed Google Scholar), and light and electron microscopic studies of PLC δ in neurofibrillary tangles suggest a role in Alzheimer's disease (9Shimohama S. Perry G. Richey P. Praprotnik D. Takenawa T. Fukami K. Whitehouse P.J. Kimura J. Brain Res. 1995; 669: 217-224Crossref PubMed Scopus (18) Google Scholar). PLC δ1 hydrolyzes substrate at a lipid/water interface. Kinetic analyses developed by Dennis and co-workers (1Hendrickson H.S. Dennis E.A. J. Biol. Chem. 1984; 259: 5734-5739Abstract Full Text PDF PubMed Google Scholar, 2Carman G.M. Deems R.A. Dennis E.A. J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar) are used to describe this type of enzymatic activity. According to this dual phospholipid binding model, a soluble enzyme must first associate with lipid (either specifically or nonspecifically) to anchor the enzyme to the lipid/water interface. Enzyme bound to the interface can then subsequently bind its substrate (within the interface) at the catalytic site where substrate hydrolysis occurs. For enzymes operating in the “scooting” or processive mode, multiple catalytic cycles can occur before the enzyme detaches from the interface (10Berg O.G. Yu B.Z. Rogers J. Jain M.K. Biochemistry. 1991; 30: 7283-7297Crossref PubMed Scopus (182) Google Scholar, 11Jain M.K. Ranadive G. Yu B.Z. Verheij H.M. Biochemistry. 1991; 30: 7330-7340Crossref PubMed Scopus (86) Google Scholar). The pleckstrin homology (PH) domain of PLC δ1 lies within the NH2-terminal region of the enzyme. Both this NH2-terminal region (12Rebecchi M. Peterson A. McLaughlin S. Biochemistry. 1992; 31: 12742-12747Crossref PubMed Scopus (175) Google Scholar, 13Cifuentes M.E. Honkanen L. Rebecchi M.J. J. Biol. Chem. 1993; 268: 11586-11593Abstract Full Text PDF PubMed Google Scholar) and the PH domain specifically (14Lomasney J.W. Cheng H.-F. Wang L.-P. Kuan Y.-S. Liu S.-M. Fesik S.W. King K. J. Biol. Chem. 1996; 271: 25316-25326Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) bind to PI(4,5)P2 with high affinity. Further, this binding is important for processive catalysis to occur (13Cifuentes M.E. Honkanen L. Rebecchi M.J. J. Biol. Chem. 1993; 268: 11586-11593Abstract Full Text PDF PubMed Google Scholar, 14Lomasney J.W. Cheng H.-F. Wang L.-P. Kuan Y.-S. Liu S.-M. Fesik S.W. King K. J. Biol. Chem. 1996; 271: 25316-25326Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). This study seeks to determine the mechanisms of PH domain-mediated activation of PLC δ1. A mutant PLC δ1 (E54K) was constructed to generate a PH domain enhancement-of-function mutant. This single amino acid substitution at position 54 of PLC δ1 dramatically increases the activity of this enzyme. Using kinetic analyses we show that changes in both the equilibrium binding constant (K s ) andV max contribute to the increased rate of processive hydrolysis, whereas the interfacial binding constant (K m ) does not contribute. The position of this substitution within the PH domain of PLC δ1 uniquely demonstrates the functional importance of this domain. Further, the increase inV max for E54K suggests a new role for PH domains as allosteric regulatory sites. We show that this PH domain-mediated change in K s and V max is a mechanism for PLC δ1 activation and propose that this may be a general mechanism for mutant enzymes in which overactivation leads to human disease. Phosphatidylinositol (PI) and phosphatidylethanolamine (PE) were from Avanti Polar Lipids. Radiolabeled phospholipids were purchased from DuPont NEN. PI(4,5)P2 was from Calbiochem. PLC δ1 monoclonal antibodies were a gift from Dr. Steven Roffler, Academia Sinica, Taiwan. All other reagents were purchased from Sigma and were of the highest grade possible. Replacement of the single glutamic acid residue at position number 54 with lysine in human PLC δ1 (E54K) was performed using the polymerase chain reaction described previously using the bacterial expression plasmid pRSETAplc (15Cheng H.-F. Jiang M.-J. Chen C.-L. Liu S.-M Wong L.-P. Lomasney J.W. King K. J. Biol. Chem. 1995; 270: 5495-5505Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The mutant, E54K, was subsequently confirmed by DNA sequence analysis. BL21(DE3)Escherichia coli (Novagen) were transformed with mutant as well as with wild-type PLC δ1 pRSETA constructs. At appropriate times, the culture was induced with 10 mmisopropyl-1-thio-β-d-galactopyranoside at 18 °C for about 8 h. The cells were collected by centrifugation and frozen at −20 °C. The remaining purification protocol was performed according to Cheng et al. (15Cheng H.-F. Jiang M.-J. Chen C.-L. Liu S.-M Wong L.-P. Lomasney J.W. King K. J. Biol. Chem. 1995; 270: 5495-5505Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) Briefly, cell pellets were ground to a paste in liquid nitrogen using a mortar and pestle. All subsequent steps were performed at 4 °C using prechilled buffers. The resulting powder was resuspended in 50 ml of sonication buffer (50 mm sodium phosphate, pH 8.0, 0.1 m KCl, 1 mm EGTA, 1 mm EDTA, and 0.1% Tween-20), and sonicated. This suspension was centrifuged at 17,000 rpm at 4 °C for 45 min. The supernatant was applied to 5 ml of Ni2+-nitrilotriacetic acid-agarose (Qiagen) in batch mode. The resin was washed 5 times with 250 ml of equilibration buffer and then five more times with 250 ml of equilibration buffer plus 15 mm imidazole. PLC was eluted with equilibration buffer plus 100 mm imidazole. Active fractions were identified using a PI hydrolysis assay and immunoblot. Active fractions were concentrated with Millipore 30,000 NMWL filters and then dialyzed against 1 liter of sonication buffer plus 10 μm phenylmethylsulfonyl fluoride (added fresh) and 1 mm dithiothreitol using Pierce dialysis cassettes. For purification to homogeneity, samples were applied to 0.5 ml of heparin-Sepharose (Pharmacia Biotech Inc.) equilibrated with sonication buffer. Columns were washed and eluted using a 150 ml of linear salt gradient from 0.1 to 0.5 mKCl in sonication buffer. Active fractions were identified using a PI hydrolysis assay and Coomassie gel staining. Measurement of PLC δ1 catalytic activity was performed according to the method of Cifuenteset al. (13Cifuentes M.E. Honkanen L. Rebecchi M.J. J. Biol. Chem. 1993; 268: 11586-11593Abstract Full Text PDF PubMed Google Scholar). Briefly, PI(4,5)P2 and3[H]PI(4,5)P2 (4.6 Ci/mmol) or PI and3[H]PI (11.0 Ci/mmol) in chloroform/methanol (2:1) were dried in Argon using an N-EVAP AF automatic thin film drier (Organomation). Final concentrations of PI(4,5)P2 were 1–325 μm, and the concentration of3[H]-PI(4,5)P2 was 0.033 μm in a final reaction volume of 30 μl. Final concentrations of PI were 10–150 μm, and 3[H]PI was 0.2 μm. Dried lipids were solubilized in a solution of dodecyl maltoside (ranging from at least 200 μm to 10 mm), 100 mm NaCl, and 20 mm HEPES, pH 7.0. The mixtures were sonicated, and the free Ca2+ was buffered using EGTA (stability constant for calcium/EGTA of 6.8 × 106m−1). Reactions were initiated by the addition of enzyme (0.888–566 ng) and carried out in a 30-μl volume containing mixed micelle substrate, enzyme, 10 μmor 1 mm free Ca2+, 100 mm NaCl, 5 mm dithiothreitol, 0.1% gelatin, and 20 mmHEPES, pH 7.0, at 30 °C. Reactions were stopped by the addition of 250 μl of 10% trichloroacetic acid and 25 μl of 20% Triton X-100. Samples were vortexed briefly and kept on ice for 10 min. Precipitates were sedimented at 10,000 × g at 4 °C for 1 min. Supernatants (280 μl each) were extracted with 1 ml of chloroform/methanol (2:1), and the aqueous phase (535 μl each), containing the 3[H]IP3 or3[H]IP product, was transferred to a scintillation vial and counted for 5 min. PLC δ1 activity was also determined using PI/deoxycholate mixed micelles. 50–90 ng of enzyme was added to a 50-μl assay buffer containing 300 μm PI, 25,000 cpm3[H]PI, 0.1% sodium deoxycholate, 50 mmHEPES, pH 7.0, 100 mm NaCl, 0.5 mg/ml bovine serum albumin, 3 mm Ca2+, and 1 mm EGTA. The reaction was terminated by adding 2 volumes of CHCl3/methanol/concentrated HCl (100:100:0.6 v/v/v) followed by 1 volume of 1 n HCl containing 5 mmEDTA. After vortexing and centrifugation at 10,000 × gfor 5 min, the aqueous phase was removed and counted. 20 μg of PI(4,5)P2 and 400 μg of PE were mixed and dried down to a thin film under N2. PE at 400 μg/ml (520 μm) had previously shown no detectable protein binding. Lipid mixtures were lyophilized for >4 h and stored at −80 °C. Before use, 1 ml of 180 mm sucrose was added to the lipid mixtures and bubbled with N2 for 5 min. One ml of 2 × binding buffer (100 mm HEPES, pH 7, 200 mm KCl, 10 mm EGTA, 10 mm EDTA) was added. The lipid mixture was centrifuged for 30 min at 2,000 × g at 4 °C. The pellet was redissolved in 1 ml of 50 mm HEPES, 100 mm KCl, 5 mm EGTA, 5 mm EDTA, and 200 μg/ml bovine serum albumin. This 1 × PI(4,5)P2/PE lipid vesicle solution was serially diluted into solution concentrations of 0.07–18 μmPI(4,5)P2 and 2.0–260 μm PE (constant mol fractions). Enzyme was added to 190 μl of the PI(4,5)P2/PE mixture and allowed to incubate at room temperature for 10 min. Enzyme/lipid mixtures were centrifuged at 4 °C, at 400,000 × g for 40 min. Supernatants were removed, and the pellet was redissolved in sodium dodecyl sulfate-protein buffer and run in a 15% acrylamide gel. The proteins were then immunoblotted. The kinetics for the native and mutant enzyme were fit to the dual phospholipid binding model originally used to describe the kinetics of phospholipase A2 (1Hendrickson H.S. Dennis E.A. J. Biol. Chem. 1984; 259: 5734-5739Abstract Full Text PDF PubMed Google Scholar, 2Carman G.M. Deems R.A. Dennis E.A. J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar) (Reaction 1). E+S⇄k−1k1ES+S⇄k−2k2ESS→k3ES+P REACTION1According to this model, enzyme first associates (via interaction between a noncatalytic site and lipid moiety) with the lipid/detergent mixed micelle to form an enzyme-mixed micelle complex. This interfacial binding is a function of the bulk lipid concentration in the mixed micelle. Thus, to determine the equilibrium dissociation constant for interfacial binding (denoted here asK s ), we measured substrate hydrolysis as a function of bulk substrate concentration while keeping the mol fraction of substrate constant (case I). Once the enzyme-mixed micelle complex forms substrate hydrolysis can then proceed at the catalytic site. In contrast to interfacial binding, interfacial catalysis is not dependent on bulk concentration of substrate but rather is dependent on the mol fraction of substrate in the lipid/mixed micelle. Therefore, to assay substrate binding to the catalytic site as well as the maximum rate of interfacial catalysis, both the interfacial Michaelis constant (denoted here as K m ) and interfacialV max were determined by measuring substrate hydrolysis with increasing mol fractions of substrate while keeping the bulk concentration of substrate constant (case II). Covarying bulk lipid concentration and mol fraction yielded a sigmoidal rateversus [substrate] curve indicative of cooperativity (case III). Data from all cases were fit to the Hill equation (16James S.R. Paterson A. Harden T.K. Downes C.P. J. Biol. Chem. 1995; 270: 11872-11881Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) (Equation 1) using the program PRISM (Graphpad). ν=Vmax·[S] nK′+[S] nEquation 1 ν = rate, V max = maximum rate, [S] = initial substrate concentration, n = Hill coefficient, and K′ is a complex association factor. For case I data, Equation 1reduces to the Henri Michaelis-Menten equation or simple rectangular hyperbola with slope, n, equal to 1. Data were fit to Equation 2 from which values of the equilibrium dissociation constant (K s ) were obtained. ν=Vmax·[S]Ks+[S]Equation 2 For case II data, a sigmoidal curve fit better than a rectangular hyperbola with slope equal to 1. Therefore, data were fit to Equation 3 using the Hill coefficient derived from case III data (16James S.R. Paterson A. Harden T.K. Downes C.P. J. Biol. Chem. 1995; 270: 11872-11881Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) andK m as well as V maxdetermined. ν=Vmax·[X] case IIInKm+[X] case IIInEquation 3 K m is the interfacial Michaelis-Menten constant, V max is the interfacialV max at a constant PI(4,5)P2concentration of 100 μm, X is the mol fraction of substrate, and n case III is the Hill coefficient derived from case III. Data from case III were fit to a sigmoidal curve according to Equation 1 where K′ in this case is defined by the intrinsic association factors of different lipid binding sites with the Hill coefficient, n, equal to the total number of binding sites. The cDNA for human PLC δ1 was mutated to substitute a lysine for glutamic acid at residue position 54 (Fig. 1 A). This residue lies in the β3/β4 loop of the PH domain of PLC δ1 (17Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Abstract Full Text PDF PubMed Scopus (529) Google Scholar). The backbone carbonyl of Glu-54 interacts with the 5 position of the inositol headgroup via a water-mediated hydrogen bond (17Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Abstract Full Text PDF PubMed Scopus (529) Google Scholar). We hypothesized that lysine substitution at this position would enhance affinity for phosphoinositides. This might occur because lysine is a fully charged species at physiological pH, and this ionic interaction would lead not only to displacement of a water molecule but also to enhanced bonding of the positively charged lysine with the negatively charged oxygen at the 5 phosphate of the inositol head group (Fig. 1 B). Purified proteins were assayed for their ability to hydrolyze PI in a sodium deoxycholate/PI mixed micelle assay. Both native and mutant enzyme showed a very similar specific activity toward hydrolysis of PI, 0.95 μmol/min/mg for PLC δ1 and 1.2 μmol/min/mg for E54K, ensuring that the substitution E54K has not globally affected enzyme structure or activity. PI(4,5)P2 hydrolysis was assayed according to case III conditions as described under “Experimental Procedures.” Substrate hydrolysis was measured simultaneously varying the bulk concentration and mol fraction of PI(4,5)P2. This was accomplished using multiple assays where the concentration of dodecyl maltoside was kept constant at 200 μm (to ensure that [detergent] was greater than the critical micellar concentration) and the PI(4,5)P2 concentration increased from 1 to 75 μm. With dodecyl maltoside as diluent, rates of substrate hydrolysis are linear over 4 min, withr 2 values ranging from 0.95 to 0.99. When solid lines were fit to both sets of data using Equation 1 from “Experimental Procedures,” the Hill coefficient of PLC δ1 was calculated to be 2.2 ± 0.2 and for E54K was calculated to be 2.0 ± 0.1 (Table I). Further, Fig.2 shows that E54K has a dramatically enhanced rate of catalysis. Thus, the single substitution in the PH domain of this PLC δ1 dramatically enhances its rate of hydrolysis but does not change the inherent cooperativity in enzyme activity.Table IComparison of PLC δ1 and E54K kinetic constants derived from activity assays according to case I, II, and IIIHill coefficient n case IIIInterfacialK m case IIK s case IV max case IIμmμmol/min/mgPLCδ12.2 ± 0.20.012 ± 0.002210 ± 484.9 ± 0.3E54K2.0 ± 0.10.034 ± 0.0185 ± 3631 ± 3PLC δ1 and E54K catalytic hydrolysis of PI(4,5)P2/dodecyl maltoside mixed micelles was carried out in 10 μmCa2+ (buffered with EGTA), 100 mm NaCl, 5 mm dithiothreitol, 0.1% gelatin, and 20 mmHEPES, pH 7.0. Kinetic constants were according to case I, II, or III as described under “Experimental Procedures.” The Hill coefficients (n) were calculated by fitting case III data to Equation 1. Both the interfacial Michaelis-Menten constant (K m ) as well as the interfacial V max were calculated by fitting case II data to Equation 3. The equilibrium dissociation constants (K s ) were calculated by fitting case I data to Equation 2. Data are means ± S.E. Open table in a new tab PLC δ1 and E54K catalytic hydrolysis of PI(4,5)P2/dodecyl maltoside mixed micelles was carried out in 10 μmCa2+ (buffered with EGTA), 100 mm NaCl, 5 mm dithiothreitol, 0.1% gelatin, and 20 mmHEPES, pH 7.0. Kinetic constants were according to case I, II, or III as described under “Experimental Procedures.” The Hill coefficients (n) were calculated by fitting case III data to Equation 1. Both the interfacial Michaelis-Menten constant (K m ) as well as the interfacial V max were calculated by fitting case II data to Equation 3. The equilibrium dissociation constants (K s ) were calculated by fitting case I data to Equation 2. Data are means ± S.E. The effect of the mutation E54K on secondary binding within the interface (K m ) was determined by assaying native and mutant activity according to case II conditions as described under “Experimental Procedures.” Multiple measurements of enzyme activity were made at a constant bulk concentration of PI(4,5)P2 (100 μm was chosen as this value is similar to the calculated K s for E54K (Table I)) and varying mol fractions of substrate (0.01–0.25 mol fraction PI(4,5)P2) using dodecyl maltoside as diluent (Fig. 3). The mol fraction of PI(4,5)P2 was not increased above 0.25 because the kinetics of PI(4,5)P2hydrolysis deviated from linearity above 0.25 (data not shown) as has been observed by other investigators (16James S.R. Paterson A. Harden T.K. Downes C.P. J. Biol. Chem. 1995; 270: 11872-11881Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Solid lines were fit to the data using Equation 3 (“Experimental Procedures”) which describes case II activity. The data fit a sigmoidal curve better than a hyperbolic one. This result has also been noted when measuring case II activity of PLC β (16James S.R. Paterson A. Harden T.K. Downes C.P. J. Biol. Chem. 1995; 270: 11872-11881Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and phospholipase A2 (1Hendrickson H.S. Dennis E.A. J. Biol. Chem. 1984; 259: 5734-5739Abstract Full Text PDF PubMed Google Scholar). For PLC δ1, K m = 0.012 ± 0.002, and for E54KK m = 0.034 ± 0.01 (Table I).V max for PLC δ1 was 4.9 ± 0.3 μmol/min/mg and 31 ± 3 μmol/min/mg for E54K (Table I). The effect of E54K on initial binding to the interface was determined by assaying enzyme activity according to case I conditions as described under “Experimental Procedures.” Multiple assays were performed with varying bulk concentrations of PI(4,5)P2 (10–325 μm) yet keeping the mol fraction of PI(4,5)P2 constant. This was achieved by increasing the bulk concentration of PI(4,5)P2(adding more lipid) while simultaneously increasing the concentration of dodecyl maltoside thus ensuring that the PI(4,5)P2 mol fraction was kept constant at 0.04. This value was chosen as it approximates the K m for E54K. Solid lines were fit to the data using Equation 2 which simply describes a rectangular hyperbola with slope equal to 1 from which values ofK s were determined. E54K was much more active than native enzyme even at relatively low substrate concentrations (Fig.4). The K s for PLC δ1 was calculated to be 210 ± 48 μm, whereas theK s for E54K was only 85 ± 36 μm(Table I). These values were within the range of constants determined previously for PLC β (16James S.R. Paterson A. Harden T.K. Downes C.P. J. Biol. Chem. 1995; 270: 11872-11881Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), PLC γ (18Jones G.A. Carpenter G. J. Biol. Chem. 1993; 268: 20845-20850Abstract Full Text PDF PubMed Google Scholar, 19Wahl M.I. Jones G.A. Nishibe S. Rhee S.G. Carpenter G. J. Biol. Chem. 1992; 267: 10447-10456Abstract Full Text PDF PubMed Google Scholar), and PLC δ1 (13Cifuentes M.E. Honkanen L. Rebecchi M.J. J. Biol. Chem. 1993; 268: 11586-11593Abstract Full Text PDF PubMed Google Scholar,14Lomasney J.W. Cheng H.-F. Wang L.-P. Kuan Y.-S. Liu S.-M. Fesik S.W. King K. J. Biol. Chem. 1996; 271: 25316-25326Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In addition to the kinetic analysis, a thermodynamic equilibrium centrifugation binding assay was done to examine further initial binding to substrate. This assay was not used quantitatively since the kinetic data were more reliable, and the equilibrium association constant K a (or1Ks) has not always provided good agreement with affinity constants determined kinetically (13Cifuentes M.E. Honkanen L. Rebecchi M.J. J. Biol. Chem. 1993; 268: 11586-11593Abstract Full Text PDF PubMed Google Scholar, 16James S.R. Paterson A. Harden T.K. Downes C.P. J. Biol. Chem. 1995; 270: 11872-11881Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). However, it was used qualitatively as another independent measure of the relative affinities of native and mutant enzyme for PI(4,5)P2/PE LUVs. E.54K was associated with LUVs at much lower concentrations of PI(4,5)P2 than is the case for PLC δ1 (Fig. 5). At about 1.1 μm PI(4,5)P2 PLC δ1 was no longer associated with LUVs, suggesting that it no longer bound to PI(4,5)P2 at this concentration. However, even at concentrations as low as 0.07 μm PI(4,5)P2 E54K still associated with the LUVs. Neither enzyme bound to PE LUVs alone over the range of PE concentrations used in this assay (data not shown). PI hydrolysis was assayed according to case III conditions described under “Experimental Procedures.” The concentration of dodecyl maltoside was kept constant at 200 μm, and the PI concentration increased from 10 to 150 μm. When solid lines were fit to both sets of data using Equation 1 from “Experimental Procedures,” the Hill coefficient of PLC δ1 was calculated to be 1.9 ± 0.2 and for E54K was calculated to be 2.0 ± 0.4 (Fig. 6). Fig. 6 shows that both PLC δ1 and E54K have similar rates of catalysis for PI in the absence of PI(4,5)P2 (V max for PLC δ1 = 15 ± 2 nmol/min/mg andV max for E54K = 18 ± 2 nmol/min/mg). Assays were also performed in the presence of 50 μmPI(4,5)P2. Hydrolysis of substrate under these conditions was still cooperative with Hill coefficients of 1.5 ± 0.3 for PLC δ1 and 1.6 ± 0.6 for E54K. In the presence of PI(4,5)P2 (Fig. 6), PLC δ1 activity was enhanced 2-fold (V max = 27 ± 3 nmol/min/mg), but E54K activity was enhanced more than 5-fold (V max = 95 ± 10 nmol/min/mg). d-IP3 inhibited PI(4,5)P2 hydrolysis for both native and mutant enzyme. Dodecyl maltoside/PI(4,5)P2 mixed micelles were assayed according to “Experimental Procedures” with constant PI(4,5)P2 (1 μm) and dodecyl maltoside (200 μm) concentrations. d-IP3 (0 to 25 μm) was added to each assay prior to addition of enzyme. Both enzymes were inhibited by d-IP3 in a dose-dependent manner (Fig. 7). The shif" @default.
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• W2003246046 title "A Single Amino Acid Substitution in the Pleckstrin Homology Domain of Phospholipase C δ1 Enhances the Rate of Substrate Hydrolysis" @default.
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