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37 5 -400. Fox, G. E., Magrum, L. J., Balch, W. E., Wolfe, R. S., and Woese, C. R. (1977), Proc. Natl. Acad. Sci. U.S.A. 74, 4532-4541. Ghisla, S., and Mayhew, S . G. (1976), Eur. J . Biochem. 63, 37 3-390. Ghisla, S., Hartman, U., Hemmerich, P., and Muller, F. (1973), Justus Liebigs Ann. Chem., 1388-1415. Gibson, Q. H., Massey, V., and Atherton, N. M. (1962), Biochem. J . 85, 369-383. Gunsalus, R. P., and Wolfe, R. S. (1977), Biochem. Biophys. Res. Commun. 76, 790-795. Gurd, F. R. N., Lawson, P. S.,Cochran, D. W., and Wenkert, E. (1971), J . Biol. Chem. 246, 3725-3730. Krebs, K. G., Heusser, D., and Wimmer, H. (1969), in Thin Layer Chromatography, Stahl, E., Ed., New York, N.Y., Springer-Verlag, pp 854-909. Johnson, L. R., and Jankowski, W. C. (1972), Carbon-13 N M R Spectra; A Collection of Assigned, Coded, and Indexed Spectra, New York, N.Y., Wiley-Interscience. Marbach, E. P., and Weil, M. H. (1967), Clin. Chem. 13,
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314-325. Massey, V., and Hemmerich, P. (1978), Biochemistry 17, 9-17. McBride, B. C., and Wolfe, R. S. (1971), Biochemistry 10, 2317-2324. Muller, F., and Massey, V. (1969), J . Biol. Chem. 244, 4007-4016. Rydon, H. N., and Smith, P. W. G. (1952), Nature (London) 169, 922-923. Spencer, R., Fischer, J., and Walsh, C. (1 976), Biochemistry 15, 1043-1053. Spencer, R., Fischer, J., and Walsh, C. (1 977), Biochemistry 16, 3586-3594. Stankovich, M. T., and Massey, V. (1976), Biochim. Biophys. Acta 452, 335-344. Taylor, C. D., and Wolfe, R. S. (1974), J . Biol. Chem. 249, 4879-4885. Tzeng, S. F., Wolfe, R. S., and Bryant, M. P. (1975a), J . Bacteriol. 121, 184- 19 1. Tzeng, S. F., Bryant, M. P., and Wolfe, R. S. (1975b), J . Bacteriol. 121, 192-196.
Ca2+ Binding to Porcine Pancreatic Phospholipase A2 and Its Function in Enzyme-Lipid Interaction? A. J. Slotboom,* E. H. J. M. Jansen, H. Vlijm, F. Pattus, P. Soares de Araujo,t and G. H. de Haas*
ABSTRACT: In addition to the Ca2+ ion bound in the active site, porcine pancreatic phospholipase A2 has been assumed to possess a second metal-ion binding locus, also specific for Ca2+, which enables the enzyme to interact with organized lipid-water interfaces at alkaline pH [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A., and de Haas, G. H. (1975), Biochemistry 14, 5387-53941. Because this interaction in the absence of Ca2+ is governed by the pK of the a-NH3+ group of the N-terminal L-Ala’ residue, the binding of this latter Ca2+ ion and its effect on the pK of the a-NH3+ group were studied. Titration studies of protons released during tryptic activation of the zymogen and of the I3C chemical shift of a 13C-enrichedN-terminal L-Ala’ residue in phospholipase A2 showed that, specifically, Ca2+ ions increase the pK of the group from 8.4 to 9.3. The pK values of the a-NH3+ groups of [D-Ala’]phospholipase A2 and DL-Ala-I-phospholipase A2, however, decreased considerably upon the addition of Ca2+.Ultraviolet difference spectroscopy suggested
that the second Ca2+ion binds close to the single Trp3 residue and a K D N 20 mM was estimated at pH 7.5. In the presence of micelles of the substrate analogue n-hexadecylphosphorylcholine, it was found by equilibrium dialysis that at pH 8 phospholipase A2 binds two Ca2+ions/mol of enzyme, whereas 1-bromo-2-octanone-inhibitedphospholipase A2 binds only one Ca2+ ion/mol of protein. Similar experiments at pH 6 revealed binding of only one Ca2+ ion/mol of native phospholipase A2, whereas no Ca2+ binding could be measured for 1-bromo-2-octanone-inhibitedphospholipase A2. Moreover, a strong synergistic effect of the micellar lipid on the binding of both Ca2+ ions was observed. Finally, the observations from microcalorimetry that at pH 10 1-bromo-2-octanone-inhibited phospholipase A2, lacking the first Ca2+-binding site, only binds to micelles in the presence of Ca2+are in good agreement with the existence of a second Ca2+ binding site on phospholipase A2.
Phospholipases A2 (EC 3.1.1.4) from different origins catalyze the specific hydrolysis of fatty acid ester bonds at the 2
position of 3-sn-phosphoglycerides. The pancreatic enzyme shows an absolute requirement for Ca2+ ions which bind in a 1:l molar ratio to the protein. Although Ba2+ and Sr2+bind with the same affinity, these metal ions are pure competitive inhibitors and no hydrolytic activity is found. Mg2+ ions do not bind at all. The unusually high specificity of phospholipase A2 for Ca2+ points to a specific function of this metal ion in the catalytic event. Spectroscopic evidence has been reported for the perturbation of histidine and tyrosine residues upon Ca2+ binding (Pieterson et al., 1974a). Active-site-directed irreversible inhibitors such as the halo ketones p-bromophenacyl
+ From the Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, “De Uithof’, Padualaan 8, Utrecht, The Netherlands. Receioed April 25, 1978. These investigations were carried out under the auspices of the Netherlands Foundation for Chemical Research (SON), with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO), and from the Fundaclo de Amparo a Pesquisa do Estado de S l o Paulo (Brazil) (P.S.d.A.). Present address: Department of Biochemistry, Institute of Chemistry, University of S l o Paulo, S l o Paulo, Brazil.
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0 1978 American Chemical Society
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BIOCHEMISTRY
bromide and 1-bromo-2-octanone specifically react with His48 and this inactivation reaction is strongly inhibited by Ca2+ (Volwerk et al., 1974). Moreover, the inactivated enzymes, though conserving their binding capacity for organized lipid-water interfaces, are no longer able to bind Ca2+. Therefore, it has been concluded that Ca2+ binds close to the active-site residue His48. The enzyme is secreted by the pancreas in a zymogen form which is activated by trypsin. It has been shown that the active center, where Ca2+ and monomeric substrate are bound and hydrolysis occurs, preexists in the zymogen (Pieterson et al., 1974b). The main difference between phospholipase A2 and its zymogen is the presence of a so-called "interface recognition site" (IRS)2 in the active enzyme (Verger et al., 1973; Pieterson et al., 19745). From spectroscopic studies and protection experiments against specific tryptic hydrolysis, it has been demonstrated that at least the rather hydrophobic N-terminal sequence of the enzyme Ala'-Leu-Trp-Gln-Phe-Arg6 is part of the IRS (van Dam-Mieras et al., 1975). This surface region, which is topographically distinct from the active center, allows the enzyme to penetrate into organized lipid-water interfaces. A consequence of this interaction is a reorganization of the active-center structure and an increase in the catalytic power of three to four orders of magnitude. It has been proposed that a functionally active IRS is stabilized by a specific ion pair between the a-NH3+ group of the N-terminal amino acid Ala' and a buried carboxylate function (van Dam-Mieras et al., 1975). Direct binding studies have led to the hypothesis that the enzyme can exist in a t least twa conformations E and E'. At neutral or slightly acidic pH, form E is present. This conformation, possessing the above-mentioned salt bridge and therefore an intact IRS, is able to interact with organized lipid-water interfaces. Form E', which predominates at alkaline pH, where the a-NH3+ group is deprotonated, lacks the ion pair and no IRS is present. This form is unable to penetrate into lipid-water interfaces. In certain kinetic studies (Verger et al., 1973; Pieterson, 1973), however, a high interface activity of the enzyme has been reported a t p H values well above the pK of the a-NH3+ group where the salt bridge would be expected to be disrupted. These studies were performed a t rather high Ca2+ concentrations, indicating the possibility of an additional metal-ion binding site on the enzyme at alkaline pH. Two hypotheses have been discussed to explain the stabilizing effect on the IRS by binding of this second Ca2+ ion (van Dam-Mieras et al., 1975): (1) The binding of Ca2+ causes an effective shielding of the salt bridge in a hydrophobic region. Such an apolar environment would shift the deprotonation of the a-NH3+ group to higher pH values, and the salt bridge would remain intact at alkaline pH. (2) The binding of Ca2+ stabilizes the conformation of the IRS even at p H values where the a-NH3+ group is deprotonated. In this hypothesis, the second Ca2+ ion is supposed to take over the stabilizing effect of the ion pair on the IRS. It is the purpose of this study to demonstrate that at alkaline pH porcine pancreatic phospholipase A2 possesses, indeed, an additional low-affinity Ca2+-binding site, distinct from the active-center locus. Binding of this second Ca2+ ion shifts the I Numbering starts from the N-terminal L-Ala residue of phospholipase A2. *Abbreviations used are: IRS, interface recognition site; AMPREC, fully c-amidinated prophospholipase Az; AMPA, fully e-amidinated phospholipase A2; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid: CMC, critical micellar concentration; NMR, nuclear magnetic EGTA, resonance; Tris, 2-amino-2-hydroxymethyl-1,3-propanediol; ethylene glycol bis(S-aminoethyl ether)-NN-tetraacetic acid.
SLOTBOOM ET A L .
pK of the N-terminal a-NH3+ group to higher values and stabilizes the enzyme form E a t alkaline pH. Experimental Section Materials and Methods. Porcine pancreatic prophospholipase A2 was purified from porcine pancreas and converted into phospholipase A2 by limited proteolysis as described by Nieuwenhuizen et al. (1 974). t-Amidinated prophospholipase A2 (AMPREC) and phospholipase A2 (AMPA) were prepared as described previously (Slotboom and de Haas, 1975). [D]- and [ L - [ ~ - ~ ~ C ] A I ~ ' ] A M andP A~ ~ - [ 3 - ' ~ C ] A l a - l AMPA containing 90% enriched I3C were prepared as described previously (Slotboom et al., 1977). n-Hexadecylphosphorylcholine was prepared as described (van DamMieras et al., 1975). 1-Bromo-2-octanone was synthesized essentially according to the procedure as described by Visser et al. (1971) and Mangold (1973). By using l-bromo-2-[214C]octanone,it was shown that upon complete inactivation of phospholipase A2 1 mol of the inhibitor/mol of enzyme was incorporated with the concomitant loss of 1 His residue. Just as for p-bromophenacyl bromide (Volwerk et al., 1974) also 1-bromo-2-octanone specifically reacts with His4*.P,adioactive 45CaC12 and [ 1 -I4C]heptanoic acid were obtained from the Radiochemical Centre, Amersham, England. Bovine trypsin was purchased from E. Merck AG, Darmstadt, Germany. All the other chemicals used were of the highest purity available. Protein concentrations for phospholipase A2 (and AMPA) and prophospholipase A2 (and AMPREC) were calculated from the absorbances with an E 2 8 0 n n , 1 ~of1 ~13.0 1 Cand m 12.3, respectively. For the N-terminally modified as well as for the 1-bromo-2-octanone blocked phospholipase A2 analogues a value of 13.0 was used. Enzyme activities were determined using the titrimetric assay procedure with egg-yolk lipoproteins as substrate (Nieuwenhuizen et al., 1974) or with short-chain lecithins (de Haas et al., 197 I ) . Phospholipid concentrations were determined by phosphorus analysis according to the Fiske and Subbarow (1925) method, as modified by Bartlett (1959). Fluorescence Measurements. Fluorescence spectra were recorded a t 25 "C with a Perkin-Elmer M P F 3 spectrofluorimeter using 1-cm cells as described previously (van DamMieras et al., 1975). Ultraviolet Difference Spectroscopy. Ultraviolet difference spectra were obtained a t 25 "C by means of a Shimadzu UV 210 double-beam spectrophotometer or an Aminco DW-2a spectrophotometer as described previously (Pieterson et al., 1974a; van Dam-hlieras et al., 1975). In order to avoid possible absorption due to the high metal ion concentrations, tandem cells (2 X 1 cm light path) were used. By using AGLA micrometer syringes a metal ion solution was titrated in the protein solution of the sample compartment and in the buffer solution of the reference compartment. The same volume of buffer was titrated in the protein solution of the referencc compartment (to correct for dilution of the chromophore) and in the buffer solution of the sample compartment. I3CNMR Spectroscopy. All I3Cspectra were obtained with a Bruker HX-360 spectrometer operating a t 90.5 MHz equipped with a variable-temperature unit (accuracy f 1 "C) (SON Facility, University of Groningen, Groningen, The Netherlands). By using quadrature detection, 1000-2000 transients were accumulated with a repetition time of 0.82 s and a spectral width of 10 000 Hz. An exponential multiplication of the free induction decay corresponding to a line broadening of 6 H z was applied to improve the signal to noise ratio. Chemical shifts were measured a t 25 "C from benzene
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Titration curves of protons released during tryptic activation of prophospholipase A2: -7 -I I I24
