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 disulphide bridges. These PLA2s have been classified into several groups based on the disulphide bond patterns found [Dennis, E. A. (1997) Trends Biochem. Sci. 22, 1-2]. To probe the effect of the various disulphide bond patterns on folding, stability and enzymatic properties, analogues of the secretory PLA2s were produced by protein engineering of porcine pancreatic PLA2. Refolding experiments indicate that small structural variations play an important 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 conformational stability. In contrast, introduction of a small insertion between positions 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 stability 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.
Janssen
M J
MJ
Department of Enzymology and Protein Engineering (CBLE, Instute of Biomembranes), Faculty of Chemistry, Utrecht University, The Netherlands.
Verheij
H M
HM
Slotboom
A J
AJ
Egmond
M R
MR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D013552:Swine
D017931:DNA Primers; D004220:Disulfides; D007527:Isoenzymes; D010741:Phospholipases A; D054467:Phospholipases A2