Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase

Y.S. Ho, P.J. Sheffield, J. Masuyama1, H. Arai1, J. Li1, J. Aoki1, K. Inoue1, U. Derewenda and Z.S. Derewenda2

Department of Molecular Physiology and Biological Physics, University of Virginia, Health Sciences Center, P.O. Box 10011, Charlottesville, VA 22906–0011, USA and 1 Department of Health Chemistry,Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo 7–3–1, Bunkyo-ku, Tokyo 113, Japan


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Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the {alpha}1 catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 Å resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 Å resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the Vmax of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.

Keywords: enzyme kinetics/mutagenesis/phospholipase A2/serine hydrolase/X-ray crystallography


    Introduction
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 Abstract
 Introduction
 Materials and methods
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Platelet activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is one of the most potent messenger phospholipids known to date. PAF is normally present in the body tissues in picomolar concentrations and is found both in the cytosol (Lynch and Hensen, 1986Go) and fluids such as blood plasma, urine and amniotic fluid (Pinckard et al., 1979Go; Cox et al., 1981Go; Billah and Johnston, 1983Go; Sanchez Crespo et al., 1983Go). The phospholipid is involved in many physiological and pathophysiological phenomena. These include activation of platelets (O'Flaherty and Wykle, 1983Go), activation of monocytes (Yasaka et al., 1982Go), asthma (Barnes et al., 1988Go), gastrointestinal ulceration (Braquet et al., 1988Go) and pregnancy (O'Neill, 1985Go; Angle et al., 1988Go). Owing to PAF's potency in cell activation, its levels must be tightly regulated.

PAF is hydrolyzed to an inactive metabolite, lyso-PAF, by a heterogeneous family of unique phospholipases A2 (PLA2) with marked specificity for PAF, called PAF acetylhydrolases (PAF-AHs). PAF-AHs inactivate PAF by hydrolyzing the ester bond at the sn-2 position of the phospholipid with concomitant release of free acetate. Several isoforms of PAF-AH have been identified; all differ substantially from other well characterized isoforms of PLA2 (Stafforini et al., 1997Go). PAF-AHs hydrolyze their substrate in a calcium-independent manner whereas most other PLA2s typically require calcium for catalysis (Dennis, 1994Go). Further, unlike secretory PLA2s, all PAF-AHs characterized to date are serine hydrolases (Derewenda and Derewenda, 1998Go). In concert with the ubiquitous distribution of PAF, PAF-AH activity has been found to be present in blood plasma (Farr et al., 1980Go; Blank et al., 1981Go; Stafforini et al., 1987aGo,bGo) blood cells (Stafforini et al., 1991Go, 1993Go), platelets (Karpouza and Vakirtzi-Lemonias, 1997Go) and body tissues (Lee et al., 1982Go; Yanoshita et al., 1988Go). To date, three of the mammalian PAF-AHs have been extensively characterized at the cDNA and protein levels, namely the extracellular blood plasma enzyme and two isoforms of intracellular cytosolic proteins.

The human lipoprotein-associated plasma isoform, which has anti-inflammatory properties, is a single-polypeptide-chain protein with a molecular weight of about 45 kDa (Stafforini et al., 1987). Site-directed mutagenesis studies have implicated Ser273, Asp296 and His351 in a putative catalytic triad (Tjoelker et al., 1995Go). Although originally the cDNA inferred amino acid sequence of the plasma PAF-AH seemed unique, it was later shown to be a distant homolog of several microbial lipases. The recently solved crystal structure of one of them, the Streptomyces exfoliatus lipase, provided evidence that all these proteins constitute a novel group within the well characterized superfamily of {alpha}/ß hydrolases (Wei et al., 1998Go), which encompasses such enzymes as the pancreatic lipase (Winkler et al., 1990Go), hymenoptera venom phospholipase A1 (Hoffman, 1994Go), bile-salt stimulated lipases (Wang et al., 1997Go; Chen et al., 1998Go) and other hydrolytic enzymes.

Hattori et al. (1995b) have isolated two intracellular, cytosolic isoforms of PAF-AH. The first, PAF-AH(II), is 40 kDa in size, shows 41% amino acid identity with that of the plasma isoform and consequently shares most of the plasma enzyme's structural features. PAF-AH(II) is myristoylated at the N-terminus, a modification unique among known lipases and phospholipases. This modification enables the protein to translocate to the membrane upon oxidant-induced shock to cells (Matsuzawa et al., 1997Go).

Another intracellular PAF-AH was isolated from bovine brain (Hattori et al., 1993Go). It is coded by two highly homologous genes, so that the respective 26 kDa polypeptide chains, {alpha}1 and {alpha}2, share 63% amino acid sequence identity. The expression of these proteins is regulated developmentally, so that the fetal brain is likely to contain {alpha}1/{alpha}2 heterodimers, while adult brain (and other tissues) may have predominantly the {alpha}2 homodimer (Manya et al., 1998Go). The protein does not exist in a monomeric form. When {alpha}1 and {alpha}2 are expressed individually in Escherichia coli, both form homodimers. We have recently reported the X-ray crystal structure of the recombinant {alpha}1-homodimer (Ho et al., 1997Go). The molecule contains a single {alpha}/ß domain of 231 residues with a central core of five-stranded ß-sheet and five helices flanking both sides of the sheet. The fold is unique among known lipases and phospholipases. The active site contains a novel variant of the classical chymotrypsin-like triad, with Ser47, His195 and Asp192.

Although all the different isoforms of PAF-AH are able to hydrolyze PAF, they vary with respect to specificity towards the acyl chain at the sn-2 position in the substrate. This variation reflects the different physiological roles played by these proteins. The human plasma isoform hydrolyzes substrates with short sn-2 chains up to six carbon atoms in length or an oxidized sn-2 chain (Stafforini et al., 1997Go). Bovine PAF-AH(II) has a slightly wider range of substrate specificity in that it will hydrolyze a phospholipid with up to nine carbon atoms at the acyl chain of the lipid at the sn-2 position (Hattori et al., 1993Go, 1995aGo,Hattori et al., bGo; Stafforini et al., 1997Go). Both proteins act primarily as scavengers of proinflammatory lipids, of which PAF is the most prominent example. When the plasma enzyme removes oxidized phospholipids from circulation, it indirectly inhibits the formation of foam cells, one of the early steps in atherosclerosis (Heery et al., 1995Go). Similarly, isoform II has been shown to protect cells from apoptosis (cell death) induced by oxidants, an effect mediated by the formation of oxidized phospholipids (Matsuzawa et al., 1997Go).

In contrast, the two catalytic subunits from isoform Ib, {alpha}1 and {alpha}2, are highly specific for PAF and effectively do not hydrolyze phospholipids with acyl chains longer than acetate in the sn-2 position. In the 1.7 Å resolution structure of the {alpha}1-homodimer an acetate ion is bound in the active site. Its presence was easily rationalized by the use of sodium acetate as a buffer in the crystallization medium. We postulated that this acetate represents the orientation of this group in the bound substrate. One of the two oxygen atoms of the acetate forms a hydrogen bond with N{varepsilon}2 of His195, while the other, occupying the oxyanion hole, is hydrogen bonded to the side chain of Asn104 (N{delta}1) and the main chain amides of Gly74 and Ser47. Its methyl group protrudes into a hydrophobic pocket made up of three methyl groups from Leu48, Thr103 and Leu194 (Figure 1Go). To probe the importance of these three amino acids for substrate specificity, we replaced each by a smaller residue using site-directed mutagenesis (L48->A, T103->S and L194->A) and studied the mutants using enzymatic assays and X-ray diffraction methods.



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Fig. 1. A ribbon diagram of the PAF-AH {alpha}1 subunit showing the position of the bound acetate (leaving group) and the locations of Leu48, Leu194 and Thr103, the three residues investigated in this report. Figure generated by MOLSCRIPT (Kraulis, 1991Go).

 

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Mutagenesis and protein expression

cDNA of wild-type PAF-AH{alpha}1 subunit was subcloned into pGEX4T1 plasmid (Pharmacia) as an EcoRI and SalI fragment. This plasmid, pGEX4T1:{alpha}1, was used as a template for site-directed mutagenesis. Mutations were done using the polymerase chain reaction (PCR) method (Saiki et al., 1988Go). The following mutagenic primers were used: L48A, 5'-CGGTGA- CTCTGCAGTCCAGCTGATGC-3'; T103S, 5'-GTTGGAA- CCAACCCAGACCACCACAATCTTCGG-3'; and L194A, 5'-GTACGATTACGCCCACTTAAGCCGTCTGGGG-3'. The PAF-AH {alpha}1 gene was amplified by PCR using the mutagenic primer and the 5' primer of pGEX4T1 with Taq polymerase (Gibco-BRL). This PCR product was then used as a mega primer with the 3' pGEX4T1 primer together with the original template in a second PCR reaction. The second PCR product is the mutated PAF-AH {alpha}1 gene, flanked by EcoRI and SalI restriction sites. The PCR product was subcloned into pGEX4T1 and positive clones were verified by DNA sequencing. Each mutant was expressed as a GST fusion in E.coli strain BL21. Bacteria were grown in 1 l of a liquid culture of Luria Broth until mid-log phase (OD600 0.4–0.6) and expression was induced by the addition of IPTG (final concentration 1 mM). Cultures were allowed to grow for an additional 4 h at 37 °C.

Protein purification and crystallization

Cell pellets were resuspended in 5 ml of lysis buffer (10 mM Tris–HCl at pH 8.0, 5 mM EDTA, 150 mM NaCl) and lysed by sonication 3–5 times for 1–2 min. Soluble protein was recovered by centrifugation at 17 000 r.p.m. for 20 min. The soluble protein was allowed to bind to a glutathione Sepharose 4B matrix for 1 h. The column was then washed with 100 ml of lysis buffer to remove non-specifically bound proteins. GST-{alpha}1 fusion protein was eluted in fractions using lysis buffer containing 10 mM glutathione at pH 7.5. The GST fusion protein was cleaved by the addition of 140 µl of thrombin (Sigma) and dialyzed at room temperature in lysis buffer to remove glutathione. After cleavage, the mixtures were passed through another glutathione Sepharose 4B column to remove the GST protein. This procedure produced protein pure enough for crystallization. The mutant proteins were crystallized under conditions similar to wild-type using the sitting drop method. A 3 µl volume of protein (5–9 mg/ml) was mixed with 3 µl of well solution (12–18% ammonium sulfate, Tris–HCl buffer at pH 6.8). Crystals of 0.4x0.3x0.4 mm were obtained after 14–20 days.

Kinetic studies

The substrate specificity of L48A, T103S and L194A mutants was analyzed using PAF and PAF analogs with propionate and butyrate moieties at the sn-2 position. Radiolabeled PAF and PAF analogs were synthesized from [3H]lysoPAF {lyso platelet activating factor (1-O-[3H]octadecyl-sn-glycero-3-phosphocholine), Amersham code No. TPK745}. The assay of PAF acetylhydrolase activity was performed as described previously (Hattori et al., 1995bGo). Short-chain sn-2 phospholipids were synthesized using the coupling method (Gupta et al., 1977Go). To a suspension of 4 µmol of labeled lyso-PAF in 0.4 ml of chloroform–pyridine (4:1, v/v), 2 mg of 4-(N,N-dimethylamino)pyridine and 40 µmol each of acid anhydride were added and stirred for 18 h. The product was extracted by the method of Bligh and Dryer (1959) under mild alkaline and acidic conditions three times each and then purified on a silica gel plate using chloroform–methanol–ammonia solution (65:25:5, v/v) as the mobile phase. Briefly, radioactive PAF or PAF analog (80 nM each) was mixed with the recombinant {alpha}1 mutant protein in 250 µl of 50 mM Tris–HCl, 5 mM EDTA, pH 7.4 and was incubated at 37°C for 30 min. At the end of the enzymatic reaction, 1.25 ml of lipid in chloroform–methanol (4:1, v/v) was added to stop the reaction. Methanol and 0.9% KCl were then added to the mixture to bring the final composition of the chloroform–methanol–KCl mixture to 1:1:0.9 (v/v). After the products had been extracted, lysoPAF was separated by TLC. Radioactivity corresponding to lysoPAF was measured by scintillation counting. For measurement of the KM and Vmax values, recombinant proteins (1 µg each) were mixed with different amounts of PAF (0.8, 4, 8, 40, 80, 400 and 800 µM), which contained [3H]acetyl-PAF (NEM). The acetate liberated from the PAF was measured as described by Hattori et al. (1993). KM and Vmax were calculated using a Lineweaver–Burk plot.

Crystallographic data collection

X-ray diffraction data were collected using two different sources. Data for L48A mutant were collected using the synchrotron source at the EMBL Outstation, Hamburg, Germany (X31 beam line). All experiments utilized crystals flash-frozen in a laminar stream of nitrogen at ~100 K. A single crystal was used and data to 2.1 Å resolution were obtained using a 1° oscillation range and a MAR-Research scanner. Data for the L194A mutant were collected to 2.3 Å resolution using an R-AXIS IV detector with Cu K{alpha} X-rays from an Enraf-Nonius rotating anode generator equipped with MSC double-focusing mirrors. The oscillation range used was 1°. Two sets of data were collected for the T103S mutant, one using the X31 beam line at the EMBL and another using the in-house rotating anode source. The resolution limit for the data set from the synchrotron source was 2.1 Å, but data were only 44% complete owing to insufficient time available for the experiment. With the rotating anode, the completeness of the data was 81%, with a resolution of 2.5 Å. The two data sets were therefore merged with a compromise resolution limit of 2.3 Å. The overall completeness of the data is 86.7%, obviously affected by the 44% completeness beyond 2.5 Å (data at low resolution were over 96% complete). The overall Rmerge based on common block to 2.5 Å was 13.1%, a value which is relatively high, but commonly observed when data from two different instruments and sources are scaled. We felt that the addition of strong, albeit incomplete, data in the refinement, will improve the quality of the resulting model. All diffraction data were indexed, merged and scaled using the HKL software (Otwinowski and Minor, 1997Go). Further details are provided in Table IGo.


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Table I. Data collection
 
Structure solution, refinement and comparisons

The structures of all three mutants were solved using phases calculated from the wild-type protein structure. In spite of the fact that the mutations did not affect the crystal contacts, the crystals of the three mutant proteins were not fully isomorphous with the wild-type. Therefore, direct calculation of Fobs Fcalc electron density maps resulted in maps with high noise, which were not very useful. In each case a rigid-body refinement protocol as implemented in X-PLOR (Brunger, 1992Go) was used to optimize the orientation and position of the model molecule. In each case, a significant improvement in the starting Rcryst and Rfree values was observed, as shown in Table IIGo. After rigid body refinement, but prior to any further crystallographic refinement, FobsFcalc and 2FobsFcalc electron density maps were computed using all reflections. The maps were inspected using `O' (Jones et al., 1991Go) and no significant features that would suggest a need to rebuild any part of the model were found. At this point, the mutated residues were introduced interactively into the density. Three cycles of positional and thermal parameter refinement using REFMAC from the CCP4 suite (Collaborative Computational Project Number 4, 1994Go) were then performed to improve the model. Water molecules were added at this point using the automated ARP procedure (Lamzin and Wilson, 1990Go). Only those waters that were more than a specified distance cut-off (2.2 Å) from a neighboring atom and that made reasonable hydrogen bonds were accepted and further evaluated using 2FobsFcalc and FobsFcalc maps in `O'. The protein model was also evaluated at this point against electron density. Subsequently, the atomic positions for each mutant were refined to convergence with REFMAC. The final Rcryst and Rfree are listed in Table IIGo. The three refined models were compared with the wild-type structure using LSQKAB (Kabsch, 1976Go) in the CCP4 suite and `O'. The volumes of the substrate binding site cavity in each mutant and in the wild-type enzyme were calculated using VOIDOO (Kleywegt and Jones, 1994Go). The probe used in the calculation was 1.2 Å in radius. The location of the cavity (the hydrophobic pocket) was specified using the coordinates of the carbon atom of the acetate molecule.


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Table II. Refinement statistics
 

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Mutagenesis and protein expression

All three mutants were expressed in E.coli with yields similar to those for the wild-type protein. The incorporation of mutations was verified at the cDNA level by direct sequencing of the genes. In each case, the expected changes were readily identifiable.

Enzyme kinetics

First, each of the mutants was analyzed for activity with PAF as the substrate. Contrary to our expectations, under these conditions all three mutants have a much lower specific activity than the wild-type enzyme. L48A and L194A each retain only 10% of their activity compared with the wild-type; T103S retained about 40% of its activity. The KM values of all of the mutants are very similar to that of the wild-type enzyme. However, the Vmax values for the mutants drop drastically, from fivefold in the T103S mutant to 46-fold in the L194A mutant (Table IIIGo). These results strongly suggest that the mutations did not affect the binding affinity of the substrate to the enzyme, but rather the rate of the catalytic step. We then analyzed substrate specificity in each mutant and the wild-type enzyme, by measuring relative activities using substrates with longer sn-2 substituents (Figure 2Go). When the wild-type protein was assayed with a PAF analog containing a propionate moiety in the sn-2 position, its specific activity dropped to <4% of its value against PAF. With the butyrate analog, the activity was barely detectable. In the case of T103S mutant, however, the propionate homolog shows ~30% of the specific activity that the mutant displays against PAF, with the value dropping only to 20% for the butyrate analog. The other two mutants also show increases in their relative activities against PAF analogs with C3 and C4 acyl chains in the sn-2 position, although not to the extent found in the T103S mutant. Because the relative activities of the mutants were low even for PAF (see above), it was not possible to determine accurately the KM and Vmax values for other substrates, where activity progressively decreased.


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Table III. Kinetic constants of wild-type and mutant PAF-AH
 


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Fig. 2. Substrate specificity of the mutant proteins was analyzed and the relative activities of the mutants toward the different substrates were measured (for details, see text). The results for the propionyl and butyryl derivatives of PAF were normalized to that of acetyl-PAF.

 
Crystallography

The refinement statistics for the three structures, including Rcryst, Rfree, root mean square deviations from ideality for bond lengths, angles and planar angles are listed in Table IIGo. The stereochemical parameters of the three models were inspected using PROCHECK (Laskowski et al., 1993Go). In all three mutants, the Ramachandran plot showed that all of the non-glycine residues were in the allowed region or in the generously allowed region. None was found in the disallowed region. As in the native protein, all three mutants showed weak electron density for residues 55–65 and the refined isotropic displacement parameters were elevated significantly in those residues. For each of the refined structures, the electron density was consistent with the mutagenesis results. Figure 3Go shows difference electron density corresponding to the mutated residue and adjacent amino acids. The solvent-accessible volume in the substrate-binding pocket in the wild-type protein is 36.4 Å3, while the figures for the mutants, L48A, T103S and L194A, are 76.5, 44.3 and 52.7 Å3, respectively.



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Fig. 3. An omit difference electron density map (FobsFcalc) covering tripeptides with the mutated residues in the central position. The tripeptides were deleted from phase calculation and the remainder of the protein was subjected to three cycles of crystallographic refinement using REFMAC to remove residual bias. The electron densities for (A) and (B) are contoured at 3{sigma} and (C) is contoured at 2{sigma}.

 
The only other significant change observed in the active site of all three mutants was a change in the {chi}1 torsional angle of the nucleophilic Ser47. In the mutants, the conformation of Ser47 is such that O{gamma} makes a proper hydrogen bond with N{varepsilon}2 of His195, in contrast to the wild-type structure in which the acetate in the active site forces Ser47 to adopt a somewhat different conformation which disrupts this crucial hydrogen bond (Figure 4Go).



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Fig. 4. Ribbon diagram showing the superposition of Ser47 and His195 between the wild-type and L48A mutant. L48A is shown in black ball-and-stick and the wild-type atoms are in gray. Hydrogen bonding distances from O{gamma} of Ser47 to N{varepsilon}2 of His195 and from O{gamma} of Ser47 to the oxygen of water146 in the mutant are shown.

 

    Discussion
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The results of the kinetic assays with propionate and butyrate derivatives of PAF support the notion that Leu48, Thr103 and Leu194 play a role in substrate recognition: each of the three mutants tested shows an enhanced relative ability to hydrolyze longer chain acyl groups from the sn-2 position. It is also interesting that the functional differences between the mutants can be rationalized based on the crystal structure of the wild-type protein in complex with acetate (Ho et al., 1997Go). In this structure, the methyl carbon of the leaving acetate is in closest contact (3.8 Å) with the methyl group of the side chain of Thr103. This side chain is rigid, with the B-values in the wild-type structure of around 12 Å2, as it is stabilized by a strong hydrogen bond between its hydroxyl group and the side chain of Arg141. An analysis of accessible surface in the active site shows that the methyl group of Thr103 is more exposed than the two terminal methyl groups on Leu194 and Leu48, both of which are approximately 4.1 Å from the acetate.

What is surprising, however, is that all three mutations severely compromise the relative activity of the enzyme, affecting in particular the catalytic rate. In search of an explanation, we examined the crystal structures of the mutants at an effective resolution of 2.3 Å or better. The three refined atomic models of the mutants were superimposed on the native protein using the {alpha}-carbons of core amino acids in the ß-sheet. This allows for the removal of any bias introduced by non-isomorphism of the crystal structures. The root mean square deviations between the wild-type structures and L48A, T103S and L194A respectively, calculated for main chain atoms, are shown in Figure 5Go. Overall, the differences fall mostly within 0.1–0.3 Å, a range which is consistent with the accuracy of atomic coordinates derived from data within the 2.1–2.3 Å resolution range. The large differences observed in all three models for the segment 55–64 is caused by the intrinsic flexibility of this fragment, which has very high B factors. Other areas, notably 15–30, 18–23, 90 and 154–176, are surface residues with higher B factors, often involved in crystal contacts. None of these effects is likely to have had any significant effects on the catalytic sites.



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Fig. 5. Root mean square deviation of the main-chain atoms for each of the three individual mutants against the wild-type protein. ß-Strands from the refined mutant structures were superimposed on the ß-strands of the wild-type structure using LSQKAB. The root mean square deviations for the main-chain atoms were obtained using the COMPAR program in CCP4.

 
The most conspicuous result of the mutagenesis is the increase of the solvent-accessible volume of the substrate-binding pocket in each of the three mutants. The extra volume in the T103S mutant extended the cavity deeper into the hydrophobic pocket and this appears to be of critical importance (see below). The extra volume in the substrate-binding site in the L194A mutant is mostly to the side of the cavity. The L48A mutant has the most significant increase in the binding-site volume. The fact that the highest activity against the propionyl derivative of PAF was identified in the T103S, where the generated space is relatively small, is at first puzzling. However, one should remember that the incoming acyl chain is bound to exhibit steric `preferences', depending on the conformation of the entire substrate and the methyl of Thr103 is probably located very close to the position of the putative incoming propionyl PAF. Why, then, is acetate-PAF the preferred substrate for the T103S mutant? An inspection of the stereochemistry of the active site shows that even after the methyl group is removed from Thr103, the ß-carbon of the residue is still within 5.3 Å of the acetate's methyl, a distance which barely allows the accommodation of propionate without serious steric hindrance. In the case of the L194A and L48A mutant, the extra space generated by mutagenesis may not be sufficient as there are other structural elements which can interfere with the extra methyl group, notably the catalytic His195, the backbone atoms of the nucleophilic elbow and Asp46.

The answer to the question of why there is a drastic drop in activity (and specifically Vmax) in all mutants is more difficult to answer. There is a possibility that small changes in the mutual disposition of the residues in the triad are responsible for the effect. At first, this seems to be a plausible explanation, particularly because each of the mutated amino acids is adjacent to one of the key catalytic residues: Leu48 is next to the nucleophilic Ser47, Leu194 is adjacent to His195 and Thr103 precedes in the sequence Asn104, a key residue in the oxyanion hole. In the L194A mutant we indeed observe a change in the position of the imidazole ring of His195, which moves about 0.5 Å into the space vacated by the side chain of Leu48. The side chain of Arg141 also moves into this space and closer to His195 than in the wild-type protein. In the L48A mutant, we observe small (<0.4 Å) changes to the backbone of the polypeptide chain in the nucleophilic elbow. However, we can detect no significant changes in the T103S mutant, other than the absence of the side chain methyl. We therefore believe that the reproducible effect of compromised catalytic activity in response to mutagenesis in the substrate-binding pocket is not due to secondary effects, which vary among the three mutants. It is more plausible that the specificity pocket affects the rate of acylation by guiding the substrate along the preferred approach line to the nucleophilic hydroxyl of Ser47. As this line of approach is disturbed, slow acylation becomes the rate-limiting step in catalysis, albeit substrate binding may not be significantly altered.

The catalytic mechanism of triad-containing serine hydrolases and their substrate-specificity has been the subject of numerous investigations. The data for serine proteinases are of less relevance, because of the different nature of the substrate. Of greater interest are the data on neutral lipases, all of which appear to hydrolyze the ester bond via a triad-involving mechanism and thioesterases, which fall into the same category. Both these groups are classified as acylhydrolases and share the same leaving group, i.e. acyl chains of varying lengths. In thioesterases, both acylation and deacylation may be the rate-limiting steps, depending on the type of enzyme and the substrate (Witkowski et al., 1992Go, 1994Go; Li et al., 1996Go). In neutral lipases, rigorous kinetic analysis of reaction pathways is often made difficult by the interfacial character of the reaction and the results are prone to assay-dependent artefacts (Ferrato et al., 1997Go). However, recent studies of staphylococcal lipases have shown, that, at least in those enzymes, acyl chain selectivity resides in the acylation step (Simons et al., 1997Go).

We conclude that by disturbing the acetyl-binding pocket we have also affected the acylation rate, since the scissile ester bond is no longer optimally oriented with respect to the active-site serine. When the three residues are mutated, neither acetyl-PAF nor propionyl-PAF can be hydrolyzed with efficiency. It is noteworthy that PAF-AH exerts its high selectivity by a synergistic action of steric control and acylation-rate control, both of which are optimized for the naturally occurring PAF.

The structure and catalytic properties of the type Ib brain intracellular PAF-AH are fully consistent with a highly specific esterase function, in contrast to interfacially adsorbed processive lipases and phospholipases. The active site is located at the bottom of a deep gorge allowing for diffusion of single molecules; there are no structural elements comparable to the `lids' found in neutral lipases or conspicuous hydrophobic patches close to the active site. Our results impact in a critical way on the understanding of the biological function of the brain PAF-AH. Enzymes whose sole function is hydrolytic degradation of a pool of molecules are not very specific. Digestive enzymes and plasma PAF-AH may serve as good examples. On the other hand, enzymes involved in regulatory mechanisms must be, by virtue of their function, highly selective with respect to their target. A suitable example is acetylcholinesterase. Vellom et al. (1993) examined the role of the amino acid residues controlling the specificity in this enzyme. The two types of cholinesterases known, acetylcholinesterase and butyrylcholinesterase, show distinct substrate specificities even though the substrate differs in length by only two carbon atoms. Two phenylalanines, F295 and F297, dictate the protein specificity. Mutating the phenylalanines to leucines, i.e. expanding the volume of the substrate-binding cavity, substantially reduces the activity of the enzyme towards acetylcholinesterase and dramatically increases the activity towards butyrylcholinesterase (Vellom et al. 1993Go). Albeit the effect of mutagenesis is not as radical as that observed in our experiments, the pattern is very similar.

If high selectivity is to be taken as a hallmark of a regulatory hydrolase, the brain PAF-AH may be no exception. The catalytic dimers associate in fetal and adult brain with another protein, the product of the LIS-1 gene, which plays an essential role in the development of cortex in mammals (Hattori et al., 1994Go). It is believed that the LIS1 protein, a member of the WD40 superfamily, is involved in the control of neuronal migration, an early step in cortex development (Reiner et al., 1995Go). A close homolog of LIS1, the product of the Aspergillus NudF gene, has been shown to control the migration of nuclei in the fungus (Xiang et al., 1995Go), suggesting a specific mechanism for the protein. Inhibitors of PAF-AH appear to interfere with cell migration, consistent with a role for PAF hydrolysis in the process (Adachi et al., 1997Go). Whether this event is upstream or downstream in the regulatory pathway remains unclear. However, our results generally support the notion of a signaling role for the catalytic subunits of the brain PAF-AH(Ib).


    Acknowledgments
 
We thank Dr Kenton Longenecker and Ms Sarah Garrard for valuable discussions. This project was supported by NIH grant NS36267. Coordinates have been deposited with the PDB; accession codes are L48A–1BWP, L194A–1BWQ and T103S–1BWR


    Notes
 
2 To whom correspondence should be addressed. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received February 20, 1999; revised April 20, 1999; accepted April 26, 1999.