Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K

Marcel J.W. Janssen, Wendy A.E.C. van de Wiel, Sigrid H.W. Beiboer, Muriel D. van Kampen, Hubertus M. Verheij1, Arend J. Slotboom2 and Maarten R. Egmond

Department of Enzymology and Protein Engineering (CBLE, Institute of Biomembranes), Faculty of Chemistry, Utrecht University, PO Box 80.054, 3508 TB Utrecht, The Netherlands 1 The paper is dedicated to Professor Bert Verheij who died in an accident on August 1, 1998


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The catalytic contribution of His48 in the active site of porcine pancreatic phospholipase A2 was examined using site-directed mutagenesis. Replacement of His48 by lysine (H48K) gives rise to a protein having a distorted lipid binding pocket. Activity of this variant drops below the detection limit which is 107-fold lower than that of the wild-type enzyme. On the other hand, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived from the preserved lipid binding properties of these variants. However, the substitutions H48Q and H48N strongly reduce the turnover number, i.e. by a factor of 105. Residual activity is totally lost after addition of a competitive inhibitor. We conclude that proper lipid binding on its own accelerates ester bond hydrolysis by a factor of 102. With the selected variants, we were also able to dissect the contribution of the hydrogen bond between Asp99 and His48 on conformational stability, being 5.2 kJ/mol. Another hydrogen bond with His48 is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol interacts with the enzyme. Its contribution to binding of the inhibitor in the presence of an interface was found to be 5.7 kJ/mol.

Keywords: active site histidine/conformational stability/inhibitor binding/porcine pancreatic phospholipase A2/site-directed mutagenesis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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The lipolytic enzyme phospholipase A2 (PLA2, EC 3.1.1.4) catalyses the hydrolysis of the 2-acyl ester linkage of 1,2-diacyl-sn-3-phosphoglycerides in a calcium-dependent reaction (Van Deenen and De Haas, 1964Go). The secretory enzymes are found in abundance in the mammalian pancreas, where they serve a digestive function, and in snake or bee venom. The importance of His48 in the active site of pancreatic PLA2s was first indicated by modification of these enzymes with p-bromophenacyl bromide (Volwerk et al., 1974Go). Furthermore, methylation of His48 (Verheij et al., 1980Go) shows a total loss of enzymatic activity while the binding of substrate or the cofactor Ca2+ remains intact. The {delta}1-nitrogen of His48 was shown to be methylated and it was concluded that this nitrogen atom acts as the proton acceptor during catalysis (Verheij et al., 1980Go). A catalytic mechanism of secretory PLA2s has been proposed which is shown in Figure 1Go. His48 and Asp99 have a function similar to that of the corresponding residues in the catalytic triad of the well-known serine proteases. In these enzymes an additional serine is the attacking nucleophile whereas in PLA2 this function is performed by a water molecule. The oxyanion hole, that stabilizes the transition state after nucleophilic attack, is formed in PLA2 by the backbone NH group of Gly30 assisted by the charge of the Ca2+ ion (Dijkstra et al., 1981Go, 1983Go). Structures further showed that the essential cofactor Ca2+ is coordinated by the two carboxylate oxygen atoms of Asp49 and three main-chain oxygen atoms from the so-called calcium binding loop (residues 25–33). Moreover, both Tyr52 and Tyr73 (Figure 1Go) are involved in an extended hydrogen bonding network connecting the active site to the {alpha}-amino group.



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Fig. 1. Schematic representation of the catalytic mechanism of secretory PLA2s and part of the hydrogen bonding network present in the crystal structure of porcine pancreatic PLA2.

 
The catalytic device of the secretory PLA2s is characterized by the Asp–His dyad, a water molecule, a calcium-binding loop and possibly other determinants that stabilize the transition state. To investigate the exact contribution of His48 to catalysis, this residue was subjected to mutagenesis. This report describes the expression, folding, purification and properties of three active site histidine variants of porcine pancreatic PLA2. His48 was replaced by lysine (H48K) mainly to impose a new charge interaction with Asp99 for possible stabilization of the enzyme. Substitutions for glutamine (H48Q) and asparagine (H48N) were chosen to minimize unfavourable steric contacts. Moreover, glutamine and asparagine can form hydrogen bonds which might take over this role from the two nitrogen atoms in the bifunctional His residue. The {varepsilon}1-nitrogen of His48 is hydrogen bonded to Asp99 and can be mimicked by the amide nitrogen atom of a glutamine residue (Leatherbarrow and Fersht, 1987Go). The {delta}1-nitrogen of His48 forms a hydrogen bond with the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol [(R)-C12-amido-PG] when present (Thunnissen et al., 1990Go) and can be mimicked by the amide nitrogen atom of an asparagine residue (Leatherbarrow and Fersht, 1987Go). The effects on conformational stability and interfacial inhibitor binding, respectively, were investigated for these active site variants of porcine pancreatic PLA2.


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 Materials and methods
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Materials

All enzymes used in DNA manipulations were obtained from New England Biolabs (Beverly, USA) except for the native Pfu DNA polymerase and its buffer which were both from Stratagene (La Jolla, USA). Enzymes were used according to the manufacturer's instructions. Synthetic oligonucleotides were ordered from Amersham Pharmacia Biotech (Roosendaal, The Netherlands). Ampicillin and acetylated trypsin were from Sigma Chemicals Co. (St Louis, USA). Isopropyl thio-ß-D-galactopyranoside (IPTG) was purchased from Boehringer (Mannheim, Germany). n-Dodecylphosphocholine (C12PN) and n-hexadecylphosphocholine (C16PN) were prepared as described previously (Van Dam-Mieras et al., 1975Go). The synthesis of 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC8PC) and of the inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol [(R)-C12-amido-PG] has been described previously (De Haas et al., 1990Go). CM-cellulose-52 (CM-52) was obtained from Whatman (Maidstone, UK). All other chemicals were of analytical grade.

Bacterial strains and plasmids

Escherichia coli strains were grown at 37°C in Luria–Bertani medium (Sambrook et al., 1989Go) and supplemented with 100 µg/ml ampicillin when plasmid maintenance was required. E.coli K12 strain DH5{alpha} (Hanahan, 1983Go) was used for all cloning. For expression, E.coli B strain Bl21(DE3) (Studier and Moffatt, 1986Go) was transformed with the appropriate vector. The plasmid pUC18 was used for the mutagenesis experiments and the plasmid pAB3 as the expression vector—a derivative of pRP265, in which the MscI–BamHI fragment is deleted after fill-in of the sticky BamHI end. Plasmid pRP265 (Phabagen Collection, Utrecht, Element number PC-V3271) is a derivative of the expression plasmid pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden) in which the polylinker GGATCCCCGGGAATTC has been replaced by the more extended polylinker GGATCCCCATGGTACCCGGGTCGACTAGTATGCATAAGCTTGAATTC. The fusion protein encoded by the pAB3 vector contains the PLA2 moiety fused through a small linker to the N-terminal 70 amino acids of the glutathione S-transferase gene of Schistosoma japonicum under the control of the inducible tac promoter (Smith and Johnson, 1988Go).

General DNA techniques

Competent bacterial cells were transformed with plasmid DNA using the CaCl2 method (Sambrook et al., 1989Go). Alkaline extraction of plasmid DNA (Birnboim and Doly, 1979Go) was followed by anion exchange chromatography on Qiagen pack 20 columns (Qiagen, Düsseldorf, Germany). DNA fragments were isolated from an agarose gel using the Qiaex DNA gel extraction kit (Qiagen, Düsseldorf, Germany). DNA sequencing was performed on double-stranded template DNA by the dideoxy chain termination method (Sanger et al., 1977Go) using a T7 polymerase sequencing kit (Amersham Pharmacia Biotech, Uppsala, Sweden).

Construction of PLA2 variants

The substitutions H48Q, H48N and H48K were introduced into the native porcine pancreatic PLA2 gene according to the QuikChange site-directed mutagenesis method (Stratagene, La Jolla, USA). Temperature cycling was performed in 0.5 ml Eppendorf tubes using a Pharmacia LKB Gene ATAQ Controller. The reaction mixture for temperature cycling contained 125 ng of both primers, 50 µM (each) deoxynucleoside triphosphate, 5–50 ng pUC18 containing the PLA2 moiety as template DNA, Pfu DNA polymerase buffer and 2.5 U native Pfu DNA polymerase in a total volume of 50 µl. The solution was overlayed with 30 µl mineral oil. The thermal profile involved a first denaturation step at 95°C for 0.5 min, followed by 16 cycles of denaturation at 95°C for 0.5 min, primer annealing at 55°C for 1 min and extension at 68°C for 6.5 min. The following 38mer oligonucleotides were used: WE1, 5'-GGTGCTGCGAGACACAGGACAATTGCTACAGAGATGCC-3', and WE2, 5'-GGCATCTCTGTAGCAATTGTCCTGTGTCTCGCAGCACC-3', for construction of the H48Q variant thereby introducing a MfeI restriction site; WE3, 5'-GGTGCTGCGAGACAAACGACAACTGCTACAGAGATGCC-3', and WE4, 5'-GGCATCTCTGTAGCAGTTGTCGTTTGTCTCGCAGCACC-3', introducing the H48N mutation; WE5, 5'-GGTGCTGCGAGACAAAAGACAACTGCTACAGAGATGCC-3', and WE6, 5'-GGCATCTCTGTAGCAGTTGTCTTTTGTCTCGCAGCACC-3', as H48K mutagenic primers. The sites of mutation in the oligonucleotide sequences are underlined. The mutagenesis resulted in the new plasmids pWE1 (H48Q), pWE3 (H48N) and pWE5 (H48K). Mutations were verified by sequencing these new constructs. The KpnI–StuI fragment of pWE1, the BamHI–StuI fragment of pWE3 and the KpnI–StuI fragment of pWE5 were subcloned into the pAB3 expression vector, resulting in the plasmids pWE2, pWE4 and pWE6, respectively.

Expression and purification of PLA2 variants

Fusion protein was obtained after transformation and expression in BL21-cells on a 10 l scale. The isolation of inclusion bodies and sulfonation of the fusion protein was carried out as described previously (Thannhauser and Scheraga, 1985Go; De Geus et al., 1987Go). Reoxidation of the protein took place in a mixture containing 2 M urea, 3 mM EDTA, 8 mM cysteine, 1 mM cystine and 10 mM borate at pH 8.7 in the dark at room temperature for a period of 20 h. The PLA2 concentration in the folding buffer was approximately 100 mg/l. For tryptic cleavage, CaCl2 was added (5 mM non-chelated) to the folding solution and the pH was checked to be 8.3. For the fusion protein containing wild-type (WT) PLA2, the tryptic cleavage can be followed by the appearance of enzymatic activity routinely measured in an egg-yolk suspension in the presence of deoxycholate (Nieuwenhuizen et al., 1974Go). To follow the cleavage reaction for the H48Q, H48N and H48K fusion proteins, which were expected to yield inactive enzymes, a test solution was made of 1 mg WT pro-PLA2 in 10 ml folding solution containing approximately 1 mg mutant fusion protein as estimated from SDS–PAGE. After addition of 100 µg acetylated trypsin, the appearance of enzymatic activity was followed over time. In parallel, tryptic cleavage of the mutant fusion protein without WT-precursor was performed on a large scale under the same conditions. When the activity of the test solution reached its maximum, the large-scale cleavage reaction was stopped by adjusting the pH of the solution to 4.5. Subsequent purification was achieved by chromatography using a CM-cellulose-52 column (200 ml) equilibrated with 5 mM NaAc at pH 4.8 and developed with a linear salt gradient of 0 to 0.75 M NaCl followed by a CM-cellulose-52 column (50 ml) at pH 6.0 in 10 mM NaAc developed with a linear salt gradient of 0 to 0.5 M NaCl. Dialysis of the protein samples was performed between chromatography steps followed by lyophilization after the last dialysis step. Final purity was checked by FPLC.

Analytical methods

Protein samples were analysed by 15% SDS–PAGE (Laemmli, 1970Go) with Coomassie brilliant blue staining. FPLC was performed using a Pharmacia system consisting of a GP-250 gradient programmer, two P-5000 pumps, a UV-1 single-path monitor and a REC-482 recorder. The samples (50 µg present in a 500 µl loop) were loaded onto Mono-Q or Mono-S HR 5/5 columns (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with 10 mM Tris–HCl pH 8.0, or 10 mM NaAc pH 6.0, respectively, and eluted with linear gradients of 0–0.25 M NaCl, or 0–0.5 M NaCl, respectively, at a flow rate of 1 ml/min. Positive ion mode ESI-MS spectra were obtained using a VG Platform II single quadrupole mass spectrometer (VG Biotech, Cheshire, UK). The samples were introduced through the electrospray interface by infusion of a 10 µl sample into a flow of acetonitrile/water (1:1, v/v) at 5 µl/min. Therefore, the sample was dissolved into the same acetonitrile/water solution containing 0.1% formic acid. Nitrogen was used as a nebulizer and curtain gas. The capillary tip was maintained at 3.6 kV and the cone voltage was 60 V. Mass calibration was performed by multiple-ion monitoring of horse–heart myoglobin signals. An adequate signal-to-noise ratio was obtained by averaging 10–15 spectra. The raw mass spectral data were processed and transformed using MassLynx software version 2.0.

Phospholipase activity assays

Routinely, PLA2 activity was determined using the egg-yolk assay as described by Nieuwenhuizen et al. (1974). Alternatively, activities were measured in a more sensitive titrimetric assay using the substrate diC8PC (10 mM) at pH 8.0 (5 mM Tris–HCL, 20 mM CaCl2, 150 mM NaCl) at 25°C. Similar results were obtained at twofold higher substrate concentration indicating that the activities measured were maximal velocities.

Calcium binding studies

Binding of Ca2+ to phospholipase A2 and various mutants was studied by ultraviolet difference spectroscopy on a Varian Cary 4E UV-VIS double beam spectrophotometer (Varian, Victoria, Australia) at pH 6.0 (50 mM NaAc and 100 mM NaCl) and 25°C as described previously (Pieterson et al., 1974Go). Binding parameters were calculated using non-linear regression analysis.

Direct binding of C12PN

The affinity of PLA2 for monomeric lipids was determined by following the increase of tryptophan fluorescence upon addition of increasing concentrations of the non-hydrolysable substrate analogue C12PN (CMC 1.3 mM). Spectra were recorded at 20°C with a Perkin-Elmer LS-5 Luminescence Spectrometer (Perkin-Elmer, Beaconsfield, UK). The excitation was performed at 280 nm and the emission spectra were recorded from 300 to 400 nm. The excitation and emission slit widths were 10 and 5 nm, respectively. Assays were performed in a buffer containing 100 mM NaAc, 50 mM CaCl2 and 100 mM NaCl at pH 6.0. Protein concentrations were 7 µM. The saturation curves obtained were fitted using non-linear regression analysis.

Competitive inhibitor binding studies

PLA2 (60 µM) was dissolved in 20 mM C16PN in presence of a buffer containing 50 mM CaCl2, 100 mM NaCl and 100 mM NaAc at pH 6.0. After recording a baseline, these solutions were subsequently titrated with a buffered stock solution (2.5 mM) of the inhibitor (R)-C12-amido-PG. UV absorption difference spectra were recorded at 25°C on a Varian Cary 4E UV-VIS double beam spectrophotometer equipped with tandem cuvettes (Varian, Victoria, Australia). Saturation curves of the free inhibitor concentration versus the UV difference signal were derived from the obtained spectra (Deveer et al., 1992Go). Binding parameters of the competitive inhibitor present in a lipid/water interface were calculated from the saturation curves using non-linear regression analysis.

Denaturation studies

Determination of conformational stability of a protein by guanidine hydrochloride (Gnd-HCl) induced denaturation has been reported previously (Pace, 1986Go; Pickersgill et al., 1991Go). Gnd-HCl was used as the unfolding reagent, because it is generally 1.5–2.5-fold more effective as a protein denaturant as compared with urea (Greene and Pace, 1974Go). Upon the reversible unfolding of ppPLA2 and its active site variants with Gnd-HCl, the fluorescence quantum yield increases about twofold, which was monitored by fluorescence spectroscopy using a Perkin-Elmer LS-5 Luminescence spectrophotometer (Perkin-Elmer, Beaconsfield, UK). The measurements were performed at 20°C using an excitation wavelength of 275 nm while the emission was measured at 305 nm. Both slit widths were 5 nm. Two protein stock solutions were prepared, one containing folded and the other containing unfolded PLA2. The former solution contained 7 µM PLA2, 2 mM EDTA and 50 mM PIPES at pH 7.0, while the latter contained, in addition, 8.25 M Gnd-HCl to denature the protein. Samples for the unfolding experiment, with varying amounts of Gnd-HCl ranging from 0 to 8.25 M, were prepared by mixing the necessary aliquots of each of these buffered PLA2 solutions. All samples were incubated overnight. Fluorescence of a solution containing 8.25 M Gnd-HCl appeared to be less than 5% of the total signal.

The free energy of unfolding at each point of the denaturation profile was calculated using the equation,

In this equation, {Delta}Gd is the free energy of unfolding, R is the gas constant, T the absolute temperature and Kd is the equilibrium constant of the unfolding reaction. The equilibrium constant Kd was calculated using the equation,

In this equation, Fn, Fd and Fobs represent the quantum yields of the native, i.e. correctly folded protein, the fully unfolded protein and the partially unfolded protein at that point of the denaturation profile, respectively. To obtain the free energy of unfolding in the absence of a denaturant, {Delta}Gd was plotted versus the Gnd-HCl concentration and fitted to the equation,

In this equation, {Delta}G0ISOdiad is the free energy of unfolding in the absence of a denaturant, which is a useful measure for the stability of the native protein, and m is the slope of the curve related to the susceptibility of the enzyme toward denaturation by the denaturant.


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Production of PLA2 variants

The enzymes were expressed as fusion proteins. As estimated from SDS–PAGE, large-scale expression of the PLA2 variants yielded about 1 g fusion protein for a 10 l culture. The PLA2 variants were folded and subsequently cleaved by trypsin treatment, producing the mature PLA2 variants, as described in Materials and methods. Purification by ion exchange chromatography yielded similar patterns for all PLA2 variants. The proteins were found to be pure by FPLC and showed one single band on SDS–PAGE gels. Routinely, expression and purification of the wild-type PLA2 yields approximately 500 mg enzyme. The purified H48Q, H48N and H48K variants were obtained in yields of 60, 15 and 25%, respectively, relative to WT PLA2.

The molecular mass of each purified, recombinant PLA2 variant was determined by ESI-MS analysis and found to be identical to the predicted value (Table IGo). As a control, the natural enzyme isolated from porcine pancreas (Nieuwenhuizen et al., 1974Go) was analysed. Here also, no significant mass differences were found between the natural enzyme and the corresponding recombinant WT enzyme.


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Table I. Molecular masses of WT PLA2 and its variants as determined by ESI-MS analysis
 
Enzymatic activity of PLA2 variants

In the egg-yolk assay no enzymatic activity could be detected for the three PLA2 variants even at very high protein concentrations. Using the diC8PC assay, we found that the turnover numbers of the H48Q and H48N variants were about 105-fold lower than that of the WT enzyme (Table IIGo). Even though this assay is 200-fold more sensitive than the egg-yolk assay, up to 4 mg of protein had to be present in an assay volume of 3 ml in order to accurately determine the turnover numbers of these PLA2 variants. Under identical conditions, the kcat value for the H48K variant was found to be 5.3x10–5 s–1 which is at least 2 orders of magnitude lower than that of the other two PLA2 variants. As can be seen from Table IIGo, the rate constant of the base-catalysed reaction (kuncat) measured in the presence of 4 mg BSA or in the absence of protein was found to be identical to that of the H48K variant. From the dramatic drop in catalytic activity of the PLA2 variants, it can be concluded that the imidazole ring of His48 in WT PLA2 is absolutely required for high enzymatic activity.


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Table II. Turnover numbers and corresponding {delta}{Delta}Gcat of WT PLA2 and its variants with the micellar substrate diC8PC (10 mM) at pH 8.0 and 25°C
 
The free energy difference of catalysis ({delta}{Delta}Gcat) as listed in Table IIGo for WT, H48Q and H48N PLA2 was calculated according to the equation,

In this equation, R is the gas constant, T the absolute temperature, while kcat and kuncat are the rate constants of the enzymatic and base-catalysed reactions, respectively.

Calcium binding studies

Asp49 is involved in the binding of the essential cofactor Ca2+ to PLA2 and it is therefore of interest for the study of the possible effects of substitution of the neighbouring His48 residue on the Ca2+ binding properties of the PLA2 variants. Table IIIGo gives values for the dissociation constants and molar UV-difference extinction coefficients determined by UV-difference spectroscopy at pH 6.0. It can be seen from Table IIIGo that substitution of His48 in PLA2 by asparagine or glutamine improves the affinity for Ca2+ ions 2–3-fold while substitution by lysine decreases the affinity about 1.5-fold. Table IIIGo also shows that the above mentioned substitutions of His48 lower the extinction coefficients of Ca2+ binding only to a small extent. From these findings it can be concluded that the overall conformation of the calcium binding loop is preserved despite the fact that the position of the Ca2+ binding ligand Asp49 is next to the site where the mutations have been made.


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Table III. Binding parameters at pH 6.0 of WT PLA2 and its variants for the monomeric substrate analogue C12PN, the cofactor Ca2+ and the strong competitive inhibitor (R)-C12-amido-PG
 
Direct binding of monomeric C12PN

The structural integrity of the PLA2 variants made was analysed by binding studies with the monomeric substrate analogue C12PN. Titrations of WT PLA2 and the H48Q or H48N variants with increasing amounts of monomeric C12PN gave rise to comparable, but small, increases in intrinsic tryptophan fluorescence. The signal was normalized to yield (FF0)/F0 and subsequently plotted as a function of the C12PN concentration. Thus a saturation curve was obtained from which the binding parameters were calculated assuming an equimolar complex between the proteins and C12PN. The maximal signals appeared to be similar for these proteins. The dissociation constants are shown in Table IIIGo. The data suggest that the C12PN binding pocket is preserved for the H48Q and H48N variants. Hardly any signal could be detected in the case of the H48K variant suggesting the presence of a distorted binding pocket in this PLA2 variant.

Competitive inhibitor binding studies

In the WT enzyme, binding of the competitive inhibitor (R)-C12-amido-PG is accompanied by the formation of a hydrogen bond between His48 and the inhibitor. Using UV absorption difference spectroscopy, direct binding studies of the competitive inhibitor in the active site of micelle-bound PLA2s were performed. It is necessary that all the enzyme is bound to the interface which could be effected for the native as well the PLA2 variants by the addition of 20 mM excess of C16PN. Subsequently, titration of the competitive inhibitor (R)-C12-amido-PG produced UV-difference spectra giving rise to absorption maxima at 279 and 284 nm. No differences in spectral shapes were noticed for the enzymes studied. Assuming an equimolar complex between the micelle-saturated protein and the inhibitor, the dissociation constants and the molar extinction coefficients were calculated from the saturation curves obtained by plotting the signal at 284 nm versus the free inhibitor concentration. The binding data need to be corrected for weak binding of C16PN monomers to the active site of PLA2. Correction takes place according to the equation (Yu et al., 1997Go),

In this equation, K*ISOdiai and K*ISOdiai(app.) are the real and apparent inhibition constants in the presence of an interface, respectively. K*ISOdiaC16PN is the dissociation constant for binding of C16PN monomers in the presence of an interface which has been reported to be 0.75 mole fraction (Jain et al., 1991Go). As can be seen from Table IIIGo, the affinity of the H48Q variant for binding of the inhibitor is 2.5-fold lower than that of the native PLA2, while that of the H48N variant is almost 4-fold higher. In contrast, the affinity of the H48K variant is more than 26-fold lower compared with that of native PLA2. The 10-fold difference in inhibitor binding for H48Q and H48N PLA2 suggests that Gln48 does not form a hydrogen bond with the inhibitor whereas Asn48 does. Table IIIGo shows that the molar extinction coefficients for the binding of the H48N and H48Q variants are comparable with that of native PLA2 while that of the H48K variant is 2-fold lower. From these inhibitor binding experiments it can be concluded that the imidazole ring of His48 is not required for binding of competitive inhibitors but can be replaced by asparagine without loss of function. The binding pockets of the H48Q and H48N variants are structurally conserved but the lysine at position 48 (H48K) is not tolerated.

Conformational stability

In wild-type PLA2, a hydrogen bond is present between Asp99 and the {varepsilon}2-nitrogen of His48 and the replacement of the active site histidine by glutamine, asparagine or lysine may affect the conformational stability of the enzyme. Therefore, we determined the thermodynamic unfolding parameters of WT PLA2 and its variants. For all the proteins, the unfolding proceeds as a single transition in a narrow Gnd-HCl concentration range, indicating that the unfolding can be described as a two-state process. The data points in the transition regions were used to quantify the stability of the proteins because here significant amounts of both folded and unfolded protein are present allowing accurate determination of {Delta}G0ISOdiad and m values (Table IVGo). For the mutants, the calculated D1/2 values ({Delta}G0ISOdiad/m) agree with the observed Gnd-HCl concentrations at the midpoint of unfolding. The data in Table IVGo indicate that the active site mutations all have a destabilizing effect on the enzyme, from which it can be concluded that His48 also plays an important structural role. The substitutions H48N and H48K affect the conformational stability considerably suggesting that the hydrogen bond with Asp99 has disappeared. Probably, this hydrogen bond is still present in the H48Q variant which only shows a small decrease in conformational stability.


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Table IV. Conformational stabilities of WT PLA2 and its variants as determined by guanidine hydrochloride induced denaturation
 

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Structural analyses of pancreatic PLA2s suggest that the two nitrogen atoms of the active site His48 serve two important but different functions (Dijkstra et al., 1981Go, 1983Go). Whereas the {varepsilon}2-nitrogen plays an important structural role, being responsible for the hydrogen bond between His48 and Asp99, the {delta}1-nitrogen fixes the catalytic water molecule and is therefore important for catalysis. Mutagenesis at position 48 of porcine pancreatic PLA2 was performed to investigate the exact contribution of His48 in catalysis. The {varepsilon}1-function was thereby mimicked by substitution for a glutamine residue and the {delta}1-function by substitution for an asparagine residue.

Replacement of His48 with glutamine or asparagine results in enzymes displaying residual activity measured on diC8PC which is 5 orders of magnitude lower than that of WT PLA2. The following evidence argues strongly against the possibility of WT contamination. First, the expression from single colonies and purifications of the PLA2 variants were performed under conditions in which cross-contamination was eliminated. Second, the WT enzyme and its variants are distinguishable on FPLC analyses. Third, the H48Q and H48N variants have comparable activities at pH 3.5 and 8.0 whereas WT activity clearly varies with pH.

Thermodynamically, the histidine residue in the WT enzyme contributes 30 kJ/mol to a total catalytic energy of 40 kJ/mol (Table IIGo). It is formally possible that the remaining catalytic energy of 10 kJ/mol in the H48Q and H48N variants comes from a non-specific site distinct from the active site. The following observations argue for catalysis at the active site. First, the activity is absent after addition of equimolar amounts of the competitive inhibitor (R)-C12-amido-PG in the assay. Second, the lipid binding pockets are structurally intact. Third, the substrate preference towards another substrate essentially parallels the WT enzyme (data not shown). Fourth, the residual activity is still 2 orders of magnitude above the non-enzymatic rate. This catalytic rate is in the range measured for `good' catalytic antibodies (Napper et al., 1987Go). The non-enzymatic rate is in good agreement with that of base-catalysed ester hydrolysis at pH 8.0 (Jencks and Carriuolo, 1961Go).

We have several lines of evidence that the active site mutants of porcine pancreatic PLA2 are structurally intact. First, the correct number of disulfide bridges has been formed as can be concluded from the correct molecular masses determined by ESI-MS analysis. Second, circular dichroism spectra of the PLA2 mutants have been recorded which are indistinguishable from that of the WT enzyme (data not shown). Third, calcium binding constants indicate that the calcium binding loop is preserved. Fourth, with the exception of the H48K mutant, the lipid binding pocket is preserved as is obvious from direct binding studies to C12PN and a competitive inhibitor.

In a study where His48 in bovine pancreatic PLA2 has been replaced by Gln, Asn and Ala, it has been reported that the residual activity displayed by the H48N variant is at least 40-fold higher as compared with that of the H48Q variant (Li and Tsai, 1993Go). The same mutations in the porcine enzyme give rise to proteins which differ in residual activity by only a factor of 2. Despite the high sequence homology between bovine and porcine PLA2, several structural differences are present (Dijkstra et al., 1983Go). One of these differences is that the N-terminus in the porcine enzyme is somewhat more open to the solvent than in the bovine enzyme leading to minor differences in the hydrogen bonding network. Since the N-terminal region is involved in binding of the monomeric substrate, these factors might cause the above mentioned difference between the bovine and porcine active site variants. The H48Q substitution has also been reported for the human synovial fluid PLA2 (Edwards et al., 1998Go). The residual activity displayed by this PLA2 variant has been shown to be 5% compared with the WT enzyme which is very surprising in the light of our results.

The calcium binding constants were determined for the three active site mutants. At pH 6.0, all lysine residues are positively charged, only a fraction of the histidine residues are positively charged and all Gln and Asn residues are uncharged. The affinity for calcium increases with decreasing positive charge at position 48. Most likely, the charge has a repulsive effect on Ca2+. The band at 240 nm in the PLA2–Ca2+ difference spectrum has been described to originate for about 50% from one or more perturbed tyrosine chromophores and, for the remainder, from a charge effect on a histidyl residue (Pieterson et al., 1974Go). From the molar UV-difference extinction coefficients listed in Table IIIGo we conclude, however, that His48 only contributes 10–20% to the difference spectrum.

The binding data of the competitive inhibitor (R)-C12-amido-PG suggest that glutamine, asparagine and lysine orient in quite different ways (Figure 2Go). Previously, X-ray crystal analysis has shown the presence of a hydrogen bond between the amide of the inhibitor and the {delta}1-nitrogen of His48 (Thunnissen et al., 1990Go). We suggest that in the variants H48Q, H48N and H48K the side chains are oriented as indicated in Figure 2Go, assuming that an amide -NH2 is a good hydrogen bond donor but cannot function as a hydrogen bond acceptor. The model explains the observed difference in inhibitor binding of the PLA2 variants. The difference in inhibitor binding energy ({delta}{Delta}Gi) between, for example, H48Q and H48N can be calculated using the equation,

In this equation, R is the gas constant, T the absolute temperature and K*ISOdiai is the interfacial inhibitor dissociation constant. The {delta}{Delta}Gi values are shown in Figure 2Go. For the H48Q and H48N variants, {delta}{Delta}Gi is 5.7 kJ/mol which is consistent with the free energy required for formation of a hydrogen bond. The results of the conformational stability experiments confirm the predicted orientation of glutamine, asparagine and lysine which is shown in Figure 3Go. Differences in {Delta}G0ISOdiad values are also shown in Figure 3Go. The structurally important hydrogen bond between Asp99 and His48 in the WT enzyme is still present in the H48Q variant and has disappeared in the H48N variant. {Delta}G0ISOdiad between these PLA2 variants is 5.2 kJ/mol corresponding to the formation of a hydrogen bond.



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Fig. 2. Thermodynamic cycle for binding of WT PLA2 and its active site histidine variants to the amide group of an acylaminophospholipid competitive inhibitor in the presence of an interface. The possible orientations of the residues Gln, Asn and Lys at position 48 are depicted.

 


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Fig. 3. Thermodynamic cycle for conformational stability of WT and its active site histidine variants. The possible orientations of the residues Gln, Asn and Lys at position 48 are depicted.

 
The main reason for substituting His48 with lysine was to investigate whether its positive charge would stabilize the protein structure by forming a salt bridge between this residue and the negatively charged Asp99. Our results imply that the positive charge is not tolerated at this position and just orients itelf in the opposite direction (Figure 2Go). The binding of substrate (analogues) in the H48K mutant is severely perturbed by steric hindrance of the bulky lysine chain, explaining the much lower residual activity than in the H48Q and H48N variants.

In summary, we have shown that His48 in the WT enzyme is essential for high catalytic turnover numbers by substitution of this residue by several other amino acid residues. Compared with base-catalysed ester hydrolysis, His48 lowers activation energy by approximately 30 kJ/mol. The remaining 10 kJ/mol is very likely caused by the remaining binding determinants which destabilize the ester bound. The thermodynamic approach suggests that the predicted orientations of the amino acid residues at position 48 are correct. The strategy applied here may also be of use for the study of other proteins.


    Acknowledgments
 
We are grateful to Mr C.Versluis, Department of Analytical Molecular Spectrometry at Utrecht University, for mass spectrometric analysis and Prof. dr. M.K.Jain, Department of Chemistry and Biochemistry at University of Delaware, for valuable discussions. S.H.W.Beiboer was financially supported by the Council for Chemical Sciences of the Netherlands Organisation for Scientific Research (CW-NWO).


    Notes
 
2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Birnboim,H.C. and Doly,J. (1979) Nucleic Acids Res., 7, 1513–1523.[Abstract]

De Geus,P., Van den Bergh,C.J., Kuipers,O.P., Verheij,H.M., Hoekstra,W.P.M. and De Haas,G.H. (1987) Nucleic Acids Res., 15, 3743–3759.[Abstract]

De Haas,G.H., Dijkman,R., Ransac,S. and Verger,R. (1990) Biochim. Biophys. Acta, 1046, 249–257.[ISI][Medline]

Deveer,A.M.T.J., Franken,P.A., Dijkman,R., Meeldijk,J., Egmond,M.R., Verheij,H.M., Verger,R. and De Haas,G.H. (1992) Biochim. Biophys. Acta, 1125, 73–81.[ISI][Medline]

Dijkstra,B.W., Kalk,K.H., Hol,W.G.J. and Drenth,J. (1981) J. Mol. Biol., 147, 97–123.[ISI][Medline]

Dijkstra,B.W., Renetseder,R., Kalk,K.H., Hol,W.G.J. and Drenth,J. (1983) J. Mol. Biol., 168, 163–179.[ISI][Medline]

Edwards,S.H., Baker,S.F. and Wilton,D.C. (1998) Biochem. Soc. Transact., 26, S239.[ISI][Medline]

Greene,R.F. and Pace,C.N. (1974) J. Biol. Chem., 249, 5388.[Abstract/Free Full Text]

Hanahan,D. (1983) J. Mol. Biol., 166, 577–585.

Jain,M.K., Tao,W., Rogers,J., Arenson,C., Eibl,H. and Yu,B.-Z. (1991) Biochemistry, 30, 10256–10268.[ISI][Medline]

Jencks,W.P. and Carriuolo,J. (1961) J. Am. Chem. Soc., 83, 1743–1750.[ISI]

Laemmli,U.K. (1970) Nature, 227, 680–685.[ISI][Medline]

Leatherbarrow,R.J. and Fersht,A.R. (1987) Biochemistry, 26, 8524–8528.[ISI][Medline]

Li,Y. and Tsai,M.-D. (1993) J. Am. Chem. Soc., 115, 8523–8526.[ISI]

Napper,A.D., Benkovic,S.J., Tramontano,A. and Lerner,R.A. (1987) Science, 237, 1041–1043.[ISI][Medline]

Nieuwenhuizen,W., Kunze,H. and De Haas,G.H. (1974) Methods Enzymol., 32, 147–154.[Medline]

Pace,C.N. (1986) Methods Enzymol., 131, 266–279.[Medline]

Pickersgill,R.W., Sumner,I.G., Collins,M.E., Warwicker,J., Perry,B., Bhat,K.M. and Goodenough,P.W. (1991) FEBS Lett., 281, 219–222.[ISI][Medline]

Pieterson,W.A., Volwerk,J.J. and De Haas,G.H. (1974) Biochemistry, 13, 1439–1445.[ISI][Medline]

Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York.

Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl Acad. Sci. USA, 74, 5463–5467.[Abstract]

Smith,B.S. and Johnson,K.S. (1988) Gene, 67, 31–40.[ISI][Medline]

Studier,F.W. and Moffatt,B.A. (1986) J. Mol. Biol., 189, 113–130.[ISI][Medline]

Thannhauser,T.W. and Scheraga,H.A. (1985) Biochemistry, 24, 7681–7688.[ISI][Medline]

Thunnissen,M.M.G.M., Ab,E., Kalk,K.H., Drenth,J., Dijkstra,B.W., Kuipers,O.P., Dijkman,R., De Haas,G.H. and Verheij,H.M. (1990) Nature, 347, 689–691.[ISI][Medline]

Van Dam-Mieras,M.C.E., Slotboom,A.J., Pieterson,W.A. and De Haas,G.H. (1975) Biochemistry, 14, 5387–5394.[ISI][Medline]

Van Deenen,L.L.M. and De Haas,G.H. (1964) Adv. Lipid Res., 2, 167–234.[ISI][Medline]

Verheij,H.M., Volwerk,J.J., Jansen,E.H.J.M., Puijk,W.C., Dijkstra,B.W., Drenth,J. and De Haas,G.H. (1980) Biochemistry, 19, 743–750.[ISI][Medline]

Volwerk,J.J., Pieterson,W.A. and De Haas,G.H. (1974) Biochemistry, 13, 1446–1454.[ISI][Medline]

Yu,B.-Z., Ghomashchi,F., Cajal,Y., Annand,R.R., Berg,O.G., Gelb,M.H. and Jain,M.K. (1997) Biochemistry, 36, 3870–3881.[ISI][Medline]

Received January 12, 1999; revised February 22, 1999; accepted March 3, 1999.