Phospholipase A2 and its zymogen from porcine pancreas. V. Interaction of phospholipase A2 and its zymogen with divalent metal ions

Abstract

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.

Calcium binding to porcine pancreatic prophospholipase A2 studied by 43Ca NMR

“…Caa+ binding to PPLA* has been studied using an equ~ibr~um gel fntration technique [4] and W difference spectrophotometry [4,5]. In these investigations, association constants in the range 2-4 X lo3 M-' were reported.…”

“…It has been shown that Ca" are essential for the catalytic properties of both the enzyme and the zymogen. Ca" binding to both forms has been studied by a variety of physicochemical techniques, showing that Ca'+ binds in a 1: 1 molar ratio with association constants in the range of l-5 X lo3 M-' [4,5]. From X,ray crystallographic data the Ca" binding site has been found to be located in a cavity of the protein surface near Aspd9 [ 61.…”

Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A2

“…Ultraviolet difference spectra were recorded on an Aminco DW 2A spectrophotometer equiped with a Midan data analyser, coupled to an Apple I1 desk-top computer. Measurements were carried out as described by Pieterson et al (1974) andDe Araujo et al (1979) with an enzyme solution of 20 pM in a buffer containing 20 mM Tris, pH 8.0, 25 mM CaCl, and 150 mM NaC1. From saturation curves, obtained with lipid monomers, a Kd value can be derived directly.…”

Crystallization and preliminary X-ray diffraction studies of piratoxin III, a D-49 phospholipase A2 from the venom of Bothrops pirajai

“…The enzymatically active PLA 2 s (D-49 PLA 2 s) hydrolyse the sn-2 ester bond of 1,2-diacyl-3-sn-phosphoglycerides. The D-49 PLA 2 s require calcium ion and are almost all at least 100 times more active when the substrate is condensed in micelles (Pieterson et al, 1974) or lamellar aggregates such as monolayers, vesicles and membranes (Slotboom et al, 1981).…”