Engineering the disulphide bond patterns of secretory phospholipases A2 into porcine pancreatic isozyme - The effects on folding, stability and enzymatic properties

Secretory phospholipases A2 (PLA2s) are small homologous proteins rich in d isulphide bridges. These PLA2s have been classified into several groups bas ed on the disulphide bond patterns found [Dennis, E. A. (1997) Trends Bioch em. Sci 22, 1-2]. To probe the effect of the various disulphide bond patter ns on folding, stability and enzymatic properties, analogues of the secreto ry PLA2s were produced by protein engineering of porcine pancreatic PLA2, R efolding experiments indicate that small structural variations play an impo rtant role in the folding of newly made PLA2 analogues. Introduction of a C -terminal extension together with disulphide bridge 50-131 gives rise to an enzyme that displays full enzymatic activity having increased conformation al stability. In contrast, introduction of a small insertion between positi ons 88 and 89 together with disulphide bridge 86-89 decreases the catalytic activity significantly, but does not change the stability. Both disulphide bridges 11-77 and 61-91 are important for the kinetic properties and stabi lity of the enzyme. Disulphide bridge 11-77, but not 61-91, was found to be essential to resist tryptic breakdown of native porcine pancreatic PLA2.