Eur. J. Biochem. 198,247-253 (1991)

0FEBS 1991 0014295691003315

Inactivation of Escherichia coli outer-membrane phospholipase A by the affinity label hexadecanesulfonyl fluoride Evidence for an active-site serine Anton J. G. HORREVOETS, Hubertus M. VERHEIJ and Gerard H. de HAAS Department of Enzymology and Protein Engineering, Centre for Biomembranes and Lipid Enzymology, State University of Utrecht, The Netherlands

(Received November 2, 1990/January 21, 1991) - EJB 90 1296

The Escherichia coli outer-membrane phospholipase A (OM PLA) is a membrane-bound acyl hydrolase with a broad substrate specificity. In order to obtain more insight into the mechanism of action of this enzyme, we designed an active-site-directed inhibitor for OM PLA on the basis of the known substrate specificity as a first step in the elucidation of the catalytic mechanism of this enzyme. The inhibitor, hexadecanesulfonyl fluoride, consists of a long hydrocarbon chain for high-affinity binding by the enzyme and a sulfonyl fluoride moiety as a reactive group. The kinetics of the inactivation of OM PLA by hexadecanesulfonyl fluoride were studied in Triton X-100 micelles. Inactivation is very fast, specific and shows the same characteristics with respect to acyl specificity, pH profile and metal ion requirement as the activity of OM PLA on substrates. Incubation of OM PLA with a stoichiometric amount of hexadecanesulfonyl fluoride leads to a total and irreversible loss of enzyme activity, resulting from the sulfonylation of Ser144. This Ser144, which we suggest to be the active-site serine of OM PLA, is part of the sequence HDSNG, whereas in the water-soluble serine proteases and lipases the structural motif GXSXG is normally encountered. On the basis of the kinetics of inactivation of OM PLA by hexadecanesulfonyl fluoride, we discuss a possible catalytic mechanism of the enzyme.

The Escherichia coli outer-membrane phospholipase A (OM PLA) is a 30-kDa protein located in the outer membrane of this Gram-negative bacterium (Bell et al., 1971). The enzyme seems dormant in its native state (Audet et al., 1974), but can be activated by perturbation of the E. coli envelope both by external agents like polymyxin B (Weiss et al., 1979) and by E. coli’s own bacteriocin release protein (Luirink et al., 1987). In vitro, the purified enzyme acts as a rather aspecific acyl hydrolase (Horrevoets et al., 1989). The amino acid sequence of OM PLA (Homma et al., 1984) shows no similarity to the published sequences of water-soluble acyl hydrolases like the digestive phospholipases and lipases. Therefore, the elucidation of the catalytic mechanism of this membranebound, non-homologous enzyme can be of general interest. OM PLA, like the digestive phospholipases Az, requires CaZf for its activity both on neutral substrates and substrates with a phosphate-containing headgroup (Horrevoets et al., 1989). The well known phospholipase Az inhibitor pbromophenacyl bromide (Volwerk et al., 1974), readily inactivates OM PLA as reported by Homma et al. (1984) and confirmed by us with the purified enzyme. However, upon inactivation of OM PLA by 14C-labeled p-bromoDhenacvlbromide. we found that at 95% inactivation of the Correspondence to H. M. Department Of Enzymology and Protein Engineering, Centre for Biomembranes and Lipid Enzymology, State University of Utrecht, postbus 80.054, 3508 TB Utrecht, The Netherlands Abbreviations. OM PLA, Escherichia coli outer-membrane phospholipase; PhMeSO,F, phenylmethanesulfonylfluoride. Enzyme. Outer-membrane phospholipase (EC 3.1 .l. -). Verheij3

enzyme, 6 - 10 p-bromophenacyl groups were covalently bound/OM PLA molecule (unpublished results). The presence of the catalytic activator Ca2+ led to an increase in inactivation rate. In the case of phospolipases Az, the binding of the essential cofactor Ca2+ results in a protection of these enzymes against inactivation by p-bromophenacyl bromide. Since these results indicate that p-bromophenacyl bromide is a rather aspecific inhibitor of OM PLA, we decided to look for more specific inhibiting compounds. The serine hydrolases, which catalyze ester hydrolysis via an acyl-enzyme intermediate (Kraut, 1977) can use different acyl-acceptors for the deacylation reaction. This enables lipases to catalyze (trans)esterification reactions, in addition to hydrolysis (Zaks and Klibanov, 1985). We have observed that the purified OM PLA can use alcohols like methanol as acyl acceptor during phospholipid degradation, leading to the formation of fatty acid methyl esters. This phenomenon has also been observed by Doi et al. (1972) using E. coli envelope fractions. OM PLA could therefore, like the lipases, belong to the class of serine hydrolases. The well-known serine-hydrolase inhibitors diisopropylfluorophosphate and phenylmethanesulfonyl fluoride (PhMeS02F)do not inactivate OM PLA (unpublished results), as is also observed in the case of lipases (Maylie et al., 1972): Therefore, we searched for an affinity label, i,e, an inhibitor for which the enzyme has a high affinity due to its structural reXmblance to the enzyme’s substrates, and which can covalently modify an active-site residue in the enzyme. We have PreviouslY shown that a substrate for OM PLA should consist of a long hydrocarbon chain and a more or less hydrophilic headgroup (Horrevoets et al., 1989). The

248 compound hexadecanesulfonyl fluoride meets these structural requirements: the inhibitor contains a long hydrocarbon chain for high-affinity binding by the enzyme, and it contains a polar sulfonyl fluoride moiety as reactive group. This compound indeed proved to be a very potent inhibitor of OM PLA. In this paper we describe the results obtained on the kinetics of inhibition of OM PLA by hexadecanesulfonyl fluoride and the identification of the modified residue.

mixture into cuvettes containing 1 ml of the assay mixture for the chromogenic assay, which contains a large excess of Triton X-100 and the substrate 2-hexadecanoylthioethane-1-phosphocholine. Progress curves did not deviate from linearity, indicating that the inactivation of OM PLA by hexadecanesulfonyl fluoride did not continue at a detectable rate during the activity assay. Kinetic analysis of active-site-directed inhibition

EXPERIMENTAL PROCEDURES

The inactivation of enzymes by affinity labels is generally assumed to proceed via a Michaelis complex, as discussed for serine hydrolases by Kraut (1977). Therefore, we analyzed the The E. coli outer-membrane phospholipase A (OM PLA) data according to Gold and Fahrney (1964), who assumed the was purified to homogeneity and freed from endogenous lipids mechanism of the sulfonylation reaction of chymotrypsin by as previously described (de Geus et al., 1936). Protein concen- PhMeS0,F to be analogous to that of acylation, i.e. to protrations were determined spectrophotometrically using a ceed via a Michaelis complex. The reaction is assumed to specific A z s o of 29.2, as determined by quantitative amino follow the course shown in Eqn (l), in which E is OM PLA, acid analysis. Fast-Flow Sepharose-Q and Sephadex LH-20 SF is the sulfonylfluoride, and E-SF is the intermediate and LH-60 were obtained from Pharmacia. The detergents Michdehs complex; the products P are sulfonyl OM PLA and C12-sulfobetain (N-dodecyl-N,N-dimethyl-l-ammonio-3-pro-hydrogen and fluoride ions. panesulfonate) and Triton X-100 were obtained from Serva. All other reagents were of analytical grade.

Materials

Hexadecanesulfonyl fluoride

Commercial hexadecanesulfonyl chloride (Aldrich) was converted to the corresponding fluoride (hexadecanesulfonyl fluoride) by refluxing in anhydrous acetone for 3 h in the presence of a tenfold molar excess of anhydrous ammonium fluoride. The mixture was filtered to remove the insoluble salts, concentrated under reduced pressure and diluted with water to destruct any unreacted hexadecanesulfonyl chloride. Hexadecanesulfonyl fluoride was subsequently extracted from the water with ether, dried, dissolved in hexane and purified on a silicic acid column in mixtures of hexane and ether. Finally, hexadecanesulfonyl fluoride was crystallized from petroleum ether (40 - 60) at 0 "C to yield small white needles (m.p. 40-41 "C). Combined gas chromatography and mass spectrometry (Kratos MS80-GCMS apparatus) showed the final product to be homogeneous and of the correct molecular mass (data not shown).

The kinetic evaluation of the sulfonylation reaction in the case where the sulfonyl fluoride is in large excess over the enzyme yields Eqn (2), which shows the relation between the observed first-order rate constant of inactivation (kl), the rate constant for the sulfonylation reaction in the Michaelis complex (k2), the concentration of the sulfonylfluoride ([SF]) and the Michaelis constant Ki.

In this approach, double-reciprocal plots of the observed firstorder rate constants of inactivation as a function of hexadecanesulfonyl fluoride concentrations will yield the rate constant ( k 2 )of sulfonylation in the Michaelis complex (from the intercept of the y axis) and the inhibitor concentration at which half-maximal rate of inactivation is observed (from slope = Ki/k2).

Chromogenic O M PLA assay

Activities of samples were assayed spectrophotometrically using 2-hexadecanoylthioethane-I-phosphocholine as substrate (de Geus et al. 1986). The assay buffer contained 50 mM Tris/HCl, 5 mM CaC12, 0.1 mM dithiobis(2-nitrobenzoic acid), 0.2 mM Triton X-100 and 0.25 mM substrate, at pH 3.3. Activities were calculated from the absorbance at 412 nm. Inactivation of O M PLA by hexadecanesulfonylfluoride

Inactivation of OM PLA was performed in a reaction buffer composed of 50 mM Tris/HCI pH 3.3, 3 mM Triton X-100 and 5 mM CaCl,, unless stated otherwise. Hexadecanesulfonyl fluoride is stable in the incubation buffer since no hydrolysis can be detected upon thin-layer chromatographic analysis after a 24-h incubation of the inhibitor in the reaction buffer. The reaction was started by the addition of hexadecanesulfonyl fluoride from a stock solution in anhydrous acetone. The rapid decrease in OM PLA activity was followed in time by injecting small aliquots (2 pl) of the incubation

Conversion o j the modfied serine residue

After inactivation by a stoichiometric amount of hexadecanesulfonyl fluoride, 4 mg (133 nmol) OM PLA was bound to a 0.5-ml column of Fast-Flow Sepharose-Q, equilibrated with reaction buffer (see above). The column was subsequently rinsed with 2 vol. of a buffer composed of 5 mM Tris/HCl pH 8.3, 3 mM Triton X-100 and 2 mM EDTA and 2 vol. 10 M urea. Subsequently the column was loaded with a solution of 10 M urea containing either 2 M 2-mercaptoethylamine or 2 M potassium thiolacetate to convert the sulfonylated serine residue into aminoethylcysteine and cysteine, respectively. Reaction was performed on the column for 40 h at room temperature and OM PLA was subsequently removed from the column with 70% formic acid. The protein was desalted on a 70-ml column of Sephadex LH 20 in 70% formic acid, dried in a Speedvac concentrator, resuspended in water and finally lyophilized. Quantitative amino acid analysis showed a recovery of (modified) protein of 60 - 90% in several independent experiments.

249 CNBr cleavage

OM PLA was dissolved in 70% formic acid at a concentration of 1 mg/ml (33.3 pM) and treated with a 200-fold excess of CNBr/methionine residue (35 mM) for 24 h at room temperature in the dark under nitrogen. The peptide mixture was freed from excess CNBr and its degradation products by drying in a Speedvac concentrator followed by lyophilization. No residual methionine residues could be detected by amino acid analysis. Isolation and identification of peptides

Peptides were purified by reversed-phase HPLC on a 10-cm Chromseph C8-HPLC column (Chrompack) with a Pharmacia LCC 500 HPLC apparatus. Lyophilized samples (20 - 50 nmol) were dissolved in 200 pl water (0.1 YOtrifluoroacetic acid), injected onto the column and eluted with linear gradients of acetonitrile in water (0.1YOtrifluoroacetic acid) at a flow rate of 0.5 ml/min. Elution profiles were monitored spectrophotometrically at 220 nm, and eluting peptides were collected manually. Recovery of the peptides was approximately 25%. The peptides were identified by amino acid analysis on an LKB 4151 Alpha-plus automatic amino acid analyzer, after 24-h hydrolysis of the peptides in constantboiling HCl in evacuated ampoules. The amino acid sequence of peptides was determined by automated Edman degradation on a gas-phase sequenator with subsequent identification of the phenylthiohydantoin derivatives by reversed-phase HPLC at the Unilever Research Laboratory (Vlaardingen, The Netherlands).

RESULTS Kinetics of inactivation of O M PLA by hexadecanesulfonylfluoridein Triton X-I00 micelles

Inactivation of OM PLA by hexadecanesulfonyl fluoride was performed in the presence of detergent micelles, which are necessary to solubilize the originally membrane-bound enzyme in an active form (de Geus et al., 1986; Horrevoets et al., 1989). Triton X-100 is an excellent inert solubilizer of OM PLA (cf. Horrevoets et al., 1989). Therefore we used this detergent to solubilize both the protein and the poorly soluble inhibitor. We performed inactivation experiments at several Triton X-I 00 concentrations under pseudo-first-order reaction conditions, i.e. at concentrations of hexadecanesulfonyl fluoride which exceeded OM PLA concentrations 25 - 500 times. Under these conditions, inactivation of OM PLA followed first-order kinetics, as shown by a linear relationship between the natural logarithm of remaining activity and reaction time. Saturation curves of the rate of inactivation as a function of hexadecanesulfonyl fluoride concentration were observed in all cases, indicating an active-site-directed mechanism of inhibition. The inactivation of enzymes by affinity labels is generally assumed to proceed via a Michaelis complex, as discussed for serine hydrolases by Kraut (1977). In this approach, as outlined in Experimental Procedures, doublereciprocal plots of the observed first-order rate constants of inactivation as a function of hexadecanesulfonyl fluoride concentrations will yield the rate constant ( k 2 )of sulfonylation in the Michaelis complex (from the intercept of the y axis) and the inhibitor concentration at which half-maximal rate of inactivation is observed (from slope = K i / k 2 ) . Resulting double-reciprocal plots for the inhibition of OM PLA by

-100

-50

50

0

100

1/ mol fraction C,, H,,SO,

-20

-10

0

10

20

30

150

F

40

50

1 I [I] (rnM-’)

Fig. 1. Influence of the sulfonylfluoride concentration on the rate of inactivation of OM P L A in Triton X-100 micelles. (A) Double-reciprocal plot of the pseudo-first-order rate constant of inactivation as a function of the mole fraction of hexadecanesulfonyl fluoride ([hexadecanesulfonyl fluoride]/{[hexadecanesulfonyl fluoride] + [Triton XIOO]}), at (0) 1.87 mM, (+) 2.84 mM, (H) 5.28 mM and ( 0 ) 10.2 mM Triton X-100, respectively, at an OM PLA concentration of 1.23 pM. (B) Double-reciprocal plot of pseudo-first-order rate constants of inactivation as a function of the concentration of the inhibitors (0)hexadecane sulfonyl fluoride (C16H33S02F)and (H) octanesulfonyl fluoride at Triton X-100 and OM PLA concentrations of 2.5 mM and 1.23 pM, respectively. For more details see Experimental Procedures

hexadecanesulfonyl fluoride are shown in Fig. 1 A. Rate constants of inactivation of OM PLA by hexadecanesulfonyl fluoride ( k 2 ) range over 0.29-0.1 s-’ (half-time of inactivation of 2.4- 6.9 s), indicating that the active-site sulfonylation proceeds at a high rate. In contrast to the approach outlined in Experimental Procedures for water-soluble enzymes and inhibitors, Kidoes not represent a true Michaelis dissociation constant in our case since all concentrations should be expressed as two-dimensional concentrations when dealing with micellar kinetics (Verger and de Haas, 1976). Since this is not yet possible, concentrations in micellar systems are usually expressed as mole fractions (Dennis, 1973). Rates of inactivation should then depend on the mole fraction of inhibitor in the detergent micelle, irrespective of the total three-dimensional concentrations of inhibitor and detergent. We observed that halfmaximal rates of inactivation were always reached at a mole fraction of hexadecanesulfonyl fluoride in the Triton X-100 micelle of 0.022, i.e. 1 molecule hexadecanesulfonyl fluoride/ 45 Triton X-100 molecules. This ‘affinity constant’ proved to

250

ioo

molar ratio C,6H3$0 *F I OM PLA

Fig. 2. Active-site titration of OM P L A by the inhibitor hexadecanesuljonylfluoride. The specific activity of OM PLA was determined with the chromogenic assay after a 2-h incubation with increasing amounts of the inhibitor hexadecanesulfonyl fluoride in a buffer composed of 50 mM Tris/HCl, 5 mM CaC12 and 3 mM Triton X100. Activities are expressed as percentages of the blank incubation (no inhibitor)

be independent of total Triton X-100 and hexadecanesulfonyl fluoride concentrations, as illustrated by a plot of first-order rate constants versus mole fraction of inhibitor (Fig. IA). Fig. 1B shows a comparison of the inactivation of OM PLA by hexadecanesulfonyl fluoride and its short-chain homologue octanesulfonyl fluoride at identical Triton X-I 00 concentration. The rate constants of sulfonylation (k,) by both inhibitors are nearly identical, but octanesulfonyl fluoride is bound by the enzyme with a much lower affinity ('Ki' at mole fraction of 0.3 as opposed to 0.022 for hexadecanesulfonyl fluoride), indicating that the high affinity of OM PLA for hexadecanesulfonyl fluoride is caused by the long hydrocarbon chain of this inhibitor. OM PLA is rapidly inactivated by hexadecanesulfonyl fluoride, even at concentrations which are comparable to the enzyme concentration, due to the high affinity of the enzyme for this inhibitor. Since hexadecanesulfonyl fluoride is stable in water (no hydrolysis detectable on TLC after 24-h incubation in reaction buffer) it is possible to perform a so-called active-site titration (Gold and Fahrney, 1964). OM PLA was incubated at different molar ratios of hexadecanesulfonyl fluoride/protein after which the remaining activity was determined. The results of this 'titration', as depicted in Fig. 2, are straightforward: an increase of the molar ratio of inhibitor/ protein results in a linear decrease of OM PLA activity, until all activity is lost at a molar ratio of 1. This proves that hexadecanesulfonyl fluoride is a very specific inhibitor of OM PLA, that completely inactivates the enzyme on a 1 : 1 molar basis. The hydrolysis of substrates by OM PLA is pH-dependent : rates are maximal at alkaline pH, whereas rates sharply decrease below pH 8 with an inflection point at approximately pH 7 (Horrevoets et al., 1989). Ideally, the rate of inactivation of OM PLA by a true active-site-directed inhibitor should exhibit the same pH profile. Therefore, we studied the influence of pH on the rate of inactivation of OM PLA by hexadecanesulfonyl fluoride. The affinity of OM PLA for the inhibitor did not change at pH values below pH 8 (data not shown). The clear effect of pH on the rate constant of inactivation ( k 2 )is shown in Fig. 3. From such a pH profile one can calculate the apparent pK, of the reaction according to Fersht and Renard (1974). Assuming that the deprotonation of one single ionizable group is responsible for this profile,

PH

Fig. 3. Influence of p H on the rate of inactivation of OM PLA by hexadecanesuljonylfluoride.Rate constants of inactivation ( k , ) were measured at different pH values upon incubation of OM PLA (3.5 pM) with hexadecanesulfonyl fluoride (C16H33S02F)in a solution containing 3 mM Triton X-100 and 3 mM CaCl,, buffered by 50 mM Tris/HCl (pH > 7.5), 50 mM Hepes (6 < pH > 7.5) or 50 mM sodium acetate (pH < 6), respectively. (m) The measured points; (-) the calculated plot for a single ionizable group with a pK of 6.75 (Fersht and Renard, 1974)

Table 1. Influence of divalent metalions on the rate of inactivation of OM PLA by hexadecanesuljonylfluoride The rate constants of inactivation ( k 2 )were determined as described in Experimental Procedures at saturating concentrations of the alkaline earth metals or in the presence of 2 mM EDTA. kre,is defined as the ratio k 2 / k 2 (EDTA). OM PLA specific activity was determined in the chromogenic OM PLA assay (Experimental Procedures), in the presence of the respective metal ions; nd = not detectable Metal ion

k2

k,,,

Specific activity

1250 320 24 19 1

U/mg 70 17 nd nd nd

S-I

0.150 0.0383 0.0029 0.0023 0.00012

Ca2 Sr2 Ba2+ Mg2+ (EDTA) +

+

the pK of this group would be 6.75. This indicates a possible involvement of a histidine residue in the inactivation reaction. OM PLA catalyzes the hydrolysis of substrates only in the presence of the activator Ca2+, although Sr2+ can replace Ca2+ resulting in a fourfold decreased specific activity of the enzyme (Horrevoets et al., 1989). Inactivation of OM PLA by hexadecanesulfonyl fluoride shows a similar influence of metal ions on the rate of inactivation. In the presence of 2 mM EDTA the inactivation proceeds at a low rate. Saturating concentrations of different metal ions accelerate the inactivation reaction to a varying extent (Table 1): most prominent is the effect of the activator CaZf which increases the inactivation rate 1250-fold. Although neither Mg2+ nor Ba2+ can replace Ca2 during substrate hydrolysis (Horrevoets et al., 1989), binding of these metal ions does result in increased inactivation rates. The affinity of the enzyme for hexadecanesulfonyl fluoride was not influenced by lowering the Ca2+ concentration or by replacement of this activator by the other alkaline earth metals, indicating that this metal ion is not essential for the binding of the inhibitor by the enzyme. A dissociation constant for Ca2+ of 0.6 mM could be determined by inactivating OM PLA at varying Ca2+ concentrations. This dissociation constant differs markedly from the +

kinetically determined Kca2+ of 15 pM in monoacyl-phospholipid micelles (Horrevoets et al., 1989). However, the incorporation of increasing amounts of the substrate analogue octadecylphosphocholine at mole fractions of 0.1 1, 0.20 and 0.33 in Triton X-100 micelles, results in a decrease of the dissociation constant for Ca2+from 0..6 mM to 0.31,0.16 and 0.075 mM, respectively. It is apparent that the presence of phospholipids in the Triton X-100 micelle results in a higher affinity of the enzyme for the metal ion.

A

0

co @l

a

Identification of the modified residue

The results described in the previous section indicate that hexadecanesulfonyl fluoride is a very specific active-site-directed inhibitor of OM PLA. Storage of OM PLA, inactivated by a stoichiometric amount of hexadecanesulfonyl fluoride, for several weeks at room temperature did not result in reactivation of the enzyme, indicating that a stable covalent linkage between the hexadecanesulfonyl moiety and OM PLA had been formed. Sulfonylfluorides have been shown to sulfonylate specifically the active-site serine in serine hydrolases as evidenced by conversion of the modified serine residue into a stable amino acid derivative (Fahrney and Gold, 1963;Gold, 1965; Maylie et al., 1972). This can be achieved via a nucleophilic substitution reaction with a mercaptide anion. Hexadecanesulfonyl-fluoride-treated OM PLA was reacted with 2-mercaptoethylamine, which resulted in the formation of 0.8 0.1 residue aminoethylcysteine/OM PLA molecule in several independent experiments, indicating that indeed a serine residue had been sulfonylated. The yield of aminoethylcysteine is in good agreement with results obtained for, e.g. PhMeS0,F-treated chymotrypsin (Gold, 1965). The results displayed in the previous section show that the sulfonylation of a serine residue completely abolishes OM PLA catalytic activity. The OM PLA sequence contains 19 serines, none of which is contained within the structural motif GXSXG as normally encountered in serine hydrolases (Brenner, 1988). Therefore we set out to identify the modified serine residue. Initial experiments with proteolytic enzymes like trypsin, chymotrypsin, papain or V8 protease showed that, in the presence of micelles, OM PLA is very resistant to proteolytic degration. Removal of detergent yielded insoluble protein aggregates, which did not dissolve in the presence of various chaotropic agents at concentrations which are compatible with proteolytic digestion. Such behaviour is often encountered in the case of membrane-bound proteins, therefore CNBr treatment is frequently used to fragment these hydrophobic proteins [see e. g. Takagaki et al. (1980)]. Since aminoethylcysteine is not stable during CNBr fragmentation of proteins (Cardinaud and Baker 1970), we chose to convert the modified serine residue into a cysteine residue with potassium thiolacetate, essentially as described for the conversion of the active-site serine of subtilisin (Neet and Koshland, 1966; Polgar and Bender, 1966). This reaction (see Experimental Procedures) resulted in the formation of 0.4 cysteine residue/molecule OM PLA, which does not contain cysteine in its native state. During CNBr digestion, a partial conversion of cysteine to cysteic acid occurred and, to prevent heterogeneity, we routinely subjected the peptide mixture to performic acid oxidation to convert cysteine completely to cysteic acid. Based on the published sequence of OM PLA (Homma et al., 1984), six peptides can be expected after CNBr cleavage (numbers in parentheses refer to residue numbers in the mature OM PLA): I (1 - 22), I1 (23 - 138), I11 (139 - 163), IV

I

ELUTION VOLUME ( mL)

0.2

0.1 0

cn N

a

0

ELUTION VOLUME ( mL)

Fig. 4. Isolation of CNBrpeptides of active-site-labeled O M P L A . (A) The elution profile of CNBr fragments of OM PLA from a column of Sephadex LH-60 (60 x 1 cm) eluted with a mixture of 88% formic acid/ethanol (1:4, by vol.) at a rate of 4 ml h - ' . The sample was dissolved in 0.4 ml 88% formic acid and applied to the column after dilution with 4 vol. ethanol. Fractions of 2.5 mi were collected and pooled as indicated. (B) The separation of peptides from pool B by reversed-phase HPLC using a linear gradient of acetonitrile in water supplemented with 0.1 % trifluoroacetic acid (for details see Experimental Procedures)

(164-192), V (193-264) and VI (265-269), respectively. To separate the two large peptides (I1 and V) from the smaller ones, the mixture was subjected to gel-filtration on a column of Sephadex LH-60 in a mixture of 88% formic acid/ethanol (1 :4, by vol.) essentially as described by Takagaki et al. (1980). The separation is depicted in Fig. 4A. The recovery of the peptides was higher than 90% as determined by quantitative amino acid analysis. Separation of the two larger peptides in the pooled gel-filtration fraction A could subsequently be achieved on an anion-exchange column (Sepharose-Q; 10 M urea, 10 mM Tris/HCI pH 8.3) since the peptides have an opposite charge of - 6 (11) and 2 (V) respectively. The smaller peptides I, I11 and IV which were unresolved in pool B after gel filtration (Fig. 4A) could be separated by reversed-phase HPLC as depicted in Fig. 4B (for details see Experimental Procedures). All peptides were then rechromatographed on HPLC to yield apparently homogenous preparations as shown by the amino acid compositions of the

+

252 detergent concentration on the maximal rate of inactivation (k2) or 'reactivity of the enzyme' might be related to the dependence of the specific activity of OM PLA on detergent or substrate concentrations (de Geus et al., 1986; Horrevoets et al., 1989). This phenomenon, i.e. a correlation between the rate constant of inactivation ( k 2 )and specific activity of the enzyme as function of amphiphile concentration, will be discussed in detail in the accompanying paper. The characteristics of the inactivation of OM PLA by hexadecanesulfonyl fluoride in Triton X-100 micelles strongly resemble the activity of OM PLA on substrates with regard to acyl specificity, pH profile and divalent metal ion requirement, showing that hexadecanesulfonyl fluoride is a very specific active-site-directed inhibitor of OM PLA, i.e. that the inactivation of OM PLA by hexadecanesulfonyl fluoride results from the labelling of an active-site residue. As discussed in the previous section, the inactivation of OM PLA by hexadecanesulfonyl fluoride results from the labeling of an active-site residue, which we have identified as Ser144. Despite the fact that OM PLA is an acyl hydrolase which can degrade monoacylglycerols, diacylglycerols and members of the Tween series in addition to (1yso)phosphoDISCUSSION lipids (Horrevoets et al., 1989), the amino acid sequence does The mechanism of inactivation of serine hydrolases by not show any similarity to the sequences of other acyl hydrosulfonylfluorides has been shown to be analogous to the acyla- lases. The kinetics of inhibition of OM PLA by hexadecation of these enzymes during substrate hydrolysis (Gold and nesulfonyl fluoride are similar to the inactivation of Fahrney, 1964). Therefore, we used a similar approach to chymotrypsin and other serine hydrolases by PhMeS02F, analyze our kinetic data. The rate constant for the sulfonyla- with respect to rate of active-site sulfonylation and pH depention reaction in the Michaelis complex (k2)was obtained from dence of inhibition. This might indicate a common mechanism double-reciprocal plots of observed first-order rate constants both of inhibition by the sulfonylfluoride and substrate hyof inactivation versus hexadecanesulfonyl fluoride concen- drolysis. The sequence around Ser144 of OM PLA is therefore trations. The high rate constants of sulfonylation (k2),ranging compared to the three classes of sequences which are normally over 0.1 -0.29 s-', are of the same order of magnitude as the observed in serine hydrolases (Brenner, 1988): those of prorate constant of sulfonylation of chymotrypsin by PhMeS02F teases, those of lipases (triacylglycerol acyl hydrolases) and those of carboxylesterases (Augusteyn et al., 1969), which are (0.052 s-'; Gold and Fahrney, 1964). The affinity of OM PLA for the inhibitor hexadeca- shown below (X stands for a variable residue). nesulfonyl fluoride cannot be expressed in terms of the Michaelis-Menten approach (dissociation constant K), since OM PLA: N H D S N G R it is clearly shown that the observed first-order rate constants Proteases: X G D S G G P XGH/Y S X G X do not depend on the total three-dimensional concentrations Lipases: S AGG of the inhibitor but on the two-dimensional concentration of Esterases: X G E the inhibitor in the Triton X-100 micelle. Therefore, we express A comparison of the sequence around the active-site serine the affinity of the enzyme for the inhibitor in terms of the mole fraction of inhibitor in the Triton X-100 micelle at which of OM PLA with the sequences of other serine hydrolases the half-maximal rate of inactivation (0.5 k 2 ) is observed, as reveals some similarity: (a) the glycine which is always obcan be deduced from double-reciprocal plots of pseudo-first- served as the second residue after the active-site serine is order rate constants versus inhibitor concentration (Fig. 1A). present; (b) an acidic residue (D) precedes the serine as in This approach makes it possible to compare both the 'reac- proteases (D) and carboxylesterases (E). The main difference tivity' of the enzyme towards the inhibitor and its affinity for between these sequences is the substitution of the glycine the inhibiting compound, as illustrated by the comparison which normally precedes the serine by a histidine in the OM between hexadecanesulfonyl fluoride and its short-chain hom- PLA sequence. For serine hydrolases, the catalytic mechanism depends ologue octanesulfonyl fluoride (cf. Fig. 1 B). These results confirmed our observations (Horrevoets et on a charge relay system composed of the residues aspartic al., 1989), that the active site of OM PLA has a high affinity acid/histidine/serine (Matthews et al., 1977; Kraut, 1977). for long-chain compounds, relative to its affinity for the short- Evidence for the proposed charge relay system in serine prochain detergent Triton X-100. The high-affinity binding of the teases was obtained predominantly from a careful analysis of inhibitor by the enzyme depends on the long hydrocarbon the three-dimensional structures of these enzymes [see, e. g., chain of hexadecanesulfonyl fluoride, a phenomenon which is Matthews et al. (1977), Bachovchin (1986), Sprang et al. also observed in the case of substrate binding by this originally (1987)]. A similar catalytic mechanism was proposed for the membrane-bound enzyme (Horrevoets et al., 1989). There- lipases (Brockerhoff, 1973), but the presence of the charge fore, sulfonylfluorides that do not contain such a long linear relay system could only recently be shown unambiguishly after hydrocarbon chain, e.g. PhMeS02F, will not inactivate OM the elucidation of the three-dimensional structures of two of PLA at a detectable rate. these enzymes, namely, human pancreatic lipase (Winkler et A striking feature of Fig. 1 A is the dependence of the rate al., 1990) and Rhizomucor miehei lipase (Brady et al., 1990). constant k z on Triton X-100 concentration. The effect of Also in the case of OM PLA, a detailed X-ray structure will

peptides which were in excellent agreement with those deduced from the published protein sequence (Homma et al., 1984). Purified peptides I, 11, IV and V were completely devoid of cysteic acid. The purified peptide 111, which corresponds to residues 139 - 163 in the mature protein, contained 0.4 mol cysteic acid/mol peptide, i.e. a ratio identical to that in the unfractionated protein. The amino acid sequence of peptide I11 was determined by automated Edman degradation to establish which of its four serine residues had been converted partly into cysteic acid (CysS0,H). The sequence of the first 21 amino acid residues of this 25-residue peptide was as follows: '39GYNHD(S/CysS0,H)NGRSDPTSRSWNRLY1sg. This amino acid sequence is identical to the published OM PLA sequence (Homma et al., 1984) except for the cysteic acid at position 144. This shows that only the serine which was originally present on position 144 of the mature protein had been partially converted to cysteic acid. On the basis of these results, we conclude that Ser144 had been sulfonylated by the active-site-directed inhibitor hexadecanesulfonyl fluoride.

253 be essential for further investigations into the architecture of the active site and its mechanism of catalysis. Although there are many similarities in the kinetics of substrate hydrolysis and inactivation by sulfonylfluorides between the OM PLA and the serine hydrolases, we cannot conclude whether OM PLA is a true member of this family of enzymes on the basis of the presented data. One question that remains to be solved is the function of the activator C a 2 + . In the catalytic mechanism of serine hydrolases there is no necessity for a divalent metal ion, contrary to the mechanism proposed for phospholipases Az (Verheij et al., 1980). In the case of lipases, more-or-less enhanced hydrolysis rates are observed in the presence of Ca2+ but this metal ion is never essential for catalytic activity [see, e.g. van Oort et al. (1989)l. Since the presence of the activator Ca2+ greatly enhances the rate of sulfonylation of the active-site serine of OM PLA by hexadecanesulfonyl fluoride, the metal ion seems to increase the reactivity (k,) of the active-site serine towards acylation. This makes a role of the ion in the deacylation process during ester hydrolysis, e. g. via sequestering the released fatty acid, less probable. The inactivation of serine hydrolases by sulfonylfluorides is proposed to occur via a mechanism similar to the acylation of these enzymes by their substrates, which is the first step in the hydrolysis reaction (Kraut, 1977). The other alkaline earth metals, which are all bound by OM PLA (Horrevoets et al., 1989), increase the rate of inactivation (cf. Table I), although only Sr2+ can replace Ca2+ during actual substrate hydrolysis. These results indicate that all these metal ions increase the reactivity of the active site, albeit to a different extent. C a Z + is frequently involved in maintaining the active conformation of proteins that do not need this metal ion for catalysis. The zinc protease thermolysin, for examples, contains no less than 4 C a Z + ions in its crystal structure (Colman et al., 1972). These bound calcium ions, which can be replaced by Sr2+and Ba", are not involved in the catalytic mechanism of thermolysin, but are essential for the high thermostability of this enzyme. A structural role for the metal ion in maintaining the active protein conformation would be a plausible explanation for a metal-ion-dependent serine hydrolase. Alternatively, the metal ion might function in the stabilization of the transition state intermediate, thereby replacing the oxy-anion hole in the serine proteases in a similar manner as proposed for C a Z +in the catalytic mechanism of the phospholipases Az (Verheij et al., 1980). Whether C a Z Cis actually present near the active site of OM PLA, or exerts its action via binding at a more distant position in the OM PLA molecule, can only be determined after resolving the threedimensional structure of the enzyme. An elucidation of the three-dimensional structure of OM PLA might be possible in the near future, since the first crystals of this non-water-soluble protein have been obtained (Gros et al., 1988). A comparison of the structure and mechanism of the membrane-bound OM PLA to the well studied serine proteases and lipases will then be possible. These studies will show whether OM PLA is a membrane-bound counterpart of the water-soluble serine acyl hydrolases, or acts via an at present unknown mechanism. The authors wish to thank P. D. van Wassenaar at the Unilever Research Laboratory (Vlaardingen, The Netherlands) for determining the amino acid sequence of peptides.

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