Conversion of pig pancreas phospholipase A2 by protein engineering into enzyme active against Escherichia coli treated with the bactericidal/permeability-increasing protein

• Published in: The Journal of biological chemistry 1991-03, Vol.266 (7), p.4162-4167
• Main Authors: WEISS, J, WRIGHT, G, BEKKERS, A. C. A. P. A, VAN DEN BERGH, C. J, VERHEIJ, H. M
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Antimicrobial Cationic Peptides; Biological and medical sciences; Blood Proteins - pharmacology; DNA Mutational Analysis; Enzymes and enzyme inhibitors; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; In Vitro Techniques; Kinetics; Membrane Proteins; Pancreas - enzymology; Phospholipases A - genetics; Phospholipases A - metabolism; Phospholipases A2; Protein Engineering; Structure-Activity Relationship; Swine
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(20)64301-0

Phospholipases A2 (PLA-2) are conserved enzymes that can vary widely in their activity toward certain biological targets. Activity of PLA-2 toward Escherichia coli treated with the bactericidal/permeability-increasing protein (BPI) of granulocytes has been detected only in "Group II" PLA-2 (lacking Cys11-Cys77) and correlates with overall basicity and the presence of a cluster of basic amino acids within a variable surface region near the NH2 terminus (including residues 6, 7, 10, 11, and 15). We now show that of five pancreatic PLA-2 ("Group I" enzymes) tested from different species of mammals, the human enzyme that is most basic both globally (pI 8.7) and locally (Arg-6, Lys-7, and Lys-10) is active toward BPI-treated E. coli (approximately 1-2% activity of the most active Group II PLA-2) whereas the other four PLA-2 are essentially inactive (less than 0.1%). The cDNA of the pig pancreatic PLA-2 (pI 6.4; Arg-6, Ser-7, Lys-10) has been modified by site-specific mutagenesis and the wild-type and mutant PLA-2 have been expressed in and purified from either E. coli or Saccharomyces cerevisiae to determine more precisely the structural determinants of PLA-2 activity toward BPI-treated E. coli. The single substitution of lysine (or arginine) for Ser-7 transformed the pig pancreatic PLA-2 into an active enzyme toward BPI-treated E. coli possessing 25-50% the activity of the human PLA-2. Additional modifications to increase global basicity (increase in net charge up to +4) caused a further (up to 2-fold) increase in activity. All mutant PLA-2 still containing Ser-7 possessed little or no activity toward BPI-treated E. coli. Changes in activity toward BPI-treated E. coli were accompanied by parallel changes in enzyme binding to this target. In contrast, substitution of lysine (or arginine) for Ser-7 caused little or no alteration of enzyme activity toward either autoclaved E. coli or egg yolk lipoproteins indicating no major effects on the catalytic properties of the PLA-2. This study demonstrates directly the role of NH2-terminal basic residues in the action of PLA-2 on BPI-treated E. coli and suggests that these properties mainly facilitate PLA-2 binding to this biological target.