The role of aspartic acid-49 (Asp-49) in the active site of porcine pancreatic phospholipase A2 was studied by recombinant DNA techniques: two mutant proteins were constructed containing either glutamic acid (Glu) or lysine (Lys) at position 49. Enzymatic characterization indicated that the presence of Asp-49 is essential for effective hydrolysis of phospholipids. Conversion of Asp-49 to either Glu or Lys strongly reduces the binding of Ca2+ ions, in particular for the lysine mutant, but the affinity for substrate analogues is hardly affected. Extensive purification of naturally occurring Lys-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein that was nearly inactive. Inhibition studies showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself has no enzymatic activity. Our results indicate that Asp-49 is essential for the catalytic action of phospholipase A2. The importance of Asp-49 was further evaluated by comparison of the primary sequences of 53 phospholipases A2 and phospholipase homologues showing that substitutions at position 49 are accompanied by structural variations of otherwise conserved residues. The occurrence of several nonconserved substitutions appeared to be a general characteristic of nonactive phospholipase A2 homologues.

The cDNA coding for the porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in E. coli. Expression of proPLA could only be obtained in the form of intracellular aggregates after fusing the 15 kDa proPLA to a large (greater than or equal to 45 kDa) bacterial peptide. The fusion protein was readily purified from cell lysates, and specifically cleaved. Cleavage of the fusion protein was achieved with either hydroxylamine (at Asn/Gly sequences in the denatured protein), or trypsin (between the pro- and the mature PLA in the renatured fusion protein). The former method releases a proPLA-like enzyme, while the latter directly yields PLA. Renaturation of the fusion protein was made possible by the use of a recently reported new S-sulphonation method. The released (pro)PLA was purified (yields of 2-3 mg/ltr of culture medium), and showed identical properties compared to native (pro)PLA.

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The cDNA coding for porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in Saccharomyces cerevisiae. Expression and secretion of proPLA could only be obtained after fusing the proPLA to the prepro sequence of the yeast a-mating factor. Upon secretion, the fusion protein was cleaved by the KEX2 protease yielding a 140-amino-acid zymogen-like form of the phospholipase A2. This protein was purified in high yield by ion-exchange chromatography. Limited proteolysis with trypsin cleaved the 'zymogen' to yield active phospholipase A2, which was indistinguishable from the authentic porcine pancreatic enzyme. These results show that a protein with a disulphide bridge content as high as 7 per 124 amino acid residues can be correctly processed by the yeast secretory apparatus.The lipolytic enzymes comprise a family of enzymes that act on insoluble aggregated substrates such as micelles or bilayer structures. Of these enzymes the extracellular phospholipases A2 from mammalian pancreas and snake venoms, have been studied in the most detail (for recent reviews see [l -31). The X-ray structure of the enzymes from porcine and bovine pancreas and from the venom of the rattlesnake Crotalus atrox, have been determined [4 -61, and a mechanism of catalysis has been proposed [7]. Phospholipases A2 are characterized by a low molecular mass (14 kDa), seven disulphide bridges and a high stability against strong acids, heat and detergents. The enzymes from pancreas are readily isolated in large amounts as proenzymes, which are naturally activated by limited tryptic digestion [2]. Whereas the proenzyme has only low activity on monomeric substrate, the mature enzyme displays full activity on both monomeric and aggregated substrates. These features make these enzymes very attractive for detailed enzymological studies in general, as well as for the study of lipolysis in particular.Protein engineering by recombinant DNA techniques has become an important tool in the study of enzymes. Therefore we have cloned the cDNA of porcine preprophospholipase A2 (preproPLA) [8], to apply these techniques to the study of phospholipase A2. This approach, however, heavily depends on the success of the expression system for the production of engineered proteins. Direct expression of the protein in Escherichia coli did not lead to the production of active pro-PLA because of incorrect formation of the disulphide bridges [8]. In contrast, the yeast S . cerevisiae is capable of both in vivo disulphide bridge formation and of efficient secretion of heterologous proteins. Therefore this yeast has been used as a host system for the expression and secretion of several mammalian enzymes [9 -121. In the present paper we describe the use of the yeast a-mating factor [13] to target recombinant prophospholipase A2 to the culture medium. The secreted protein was isolated from the supernatant in high yield, and, after tryptic activation, was indistinguishable from native procine pancreatic phospholipase A2. EXPERIMENTAL PROCEDUREStrains and cultu...