The localization of the previously postulated interface recognition site (IRS) in porcine pancreatic phospholipase A2, required for a specific interaction between the enzyme and organized lipid-water interfaces, was investigated by ultraviolet difference spectroscopy, by measurements of the intrinsic fluorescence of the unique Trp residue, and by protection experiments against specific tryptic hydrolysis. Using the enzymically nondegradable substrate analogues: CnH(2n+1)(0-)OOCH2CH2N+(CH3)3-(H,OH), it is shown that the rather hydrophobic N-terminal sequence of the enzyme, viz., Ala-Leu-Trp-Gln-Phe-Arg, is directly involved in the interaction with the lipid-water interface. Besides hydrophobic probably also polar interactions contribute to the binding process. At neutral or acidic pH the presence of a salt bridge between the N-terminal alpha-NH3+ group and a negatively charged side chain stablizes the interface recognition site and allows the enzyme to penetrate micellar surfaces, even in the absence of metal ion. At alkaline pH, interaction of the enzyme with micellar interfaces requires the presence of Ca2+ (Ba2+) ions.
Porcine pancreatic phospholipase Az as well as its zymogen bind calcium ions in a 1:l molar ratio at calcium concentrations ranging from 5 X to 5 X 10-5 M. Binding of calcium to both the zymogen and the active enzyme changes the protein absorption spectrum. The ultraviolet difference spectrum is characterized by a strong band at 242 nm and two smaller bands at 282 and 288 nm. The origin of the difference spectrum can be understood as a partial shielding of a tyrosine residue from the aqueous solvent and in addition, most likely, a charge effect on a histidine residue. Calcium does not influence the tryptophan fluorescence of either protein. However, addition of calcium enhances the 8-anilino-l -naphthalenesulfonate fluorescence induced by phospholipase A and its precursor. Besides the spectral changes binding of calcium protects the enzyme against inactivation by p-bromophenacyl P hospholipase AP (EC 3.1.1.4) catalyzes the hydrolysis of fatty acid ester bonds at the 2 position of 1,2-diacyl sn-phosphoglycerides (van Deenen and de Haas, 1964). The stimulating effect of calcium ions on the hydrolysis of phospholipids by phospholipase Az has long been recognized (Hayashi and Kornberg, 1954). The requirement of Ca2+ seems to be a ~~ ~ The present investigations were carried out under the auspices of The Netherlands Foundation for Chemical Research (SON) and with financial aid from The Netherlands Organization for the Advancement of Pure Research (ZWO). This is paper V in a series of studies on phospholipase AZ and its zymogen from porcine pancreas.$ Present address: Diosynth BV, Kloosterstraat 6, Oss, The Netherlands.bromide and diminishes its susceptibility to trypsin attack. These criteria suggest that the enzyme and the zymogen undergo a conformational change upon calcium binding. Substitution of calcium by barium or strontium results in a similar but somewhat smaller conformational change, in agreement with the behavior of these ions as competitive inhibitors. Magnesium, on the contrary, does not bind to the enzyme according to both kinetic and direct binding experiments. The dissociation constants of the various enzyme and zymogen metal complexes have been determined. Values were found ranging from 10-I M at pH 4.0 to 2 X M at pH 10.0. The data show a good agreement on using the various techniques and suggest that the metal ion binding site contains one or more carboxylates with an additional contribution of a residue with a pKof 6-7, presumably histidine.
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