Probing the Mechanism of Pancreatic Phospholipase A2 with the Aid of Recombinant DNA Techniques

Biochemistry and Physiology of Mammalian Secreted Phospholipases A2

“…The originally proposed active site catalytic mechanism for hydrolysis of the sn-2 ester of phospholipids by sPLA2s is shown in Figure 4a,b (35). It is based on highresolution X-ray structures of several sPLA2s and the conservation of structural features of the active site, including an Asp-His catalytic diad and a Ca 2+ ion bound by a peptide loop.…”

INTERFACIAL ENZYMOLOGY OF GLYCEROLIPID HYDROLASES: Lessons from Secreted Phospholipases A2

“…The roles of sPLAzs in signal transduction and inflammatory processes have been extensively investigated (for reviews from 1990 onward, see [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. Previous reviews on the in vitro properties of purified sPLAzs include those on structure/function (9,21,25,27,(46)(47)(48)(49), interfacial recognition (50, 51), interfacial kinetics (4, 18, 52-56), and inhibition (32,(57)(58)(59)(60)(61). Elements of these reviews, structural studies, and more recent data that relate to the fu n-damental concepts of interfacial catalysis described in the first part of the present review are summarized in this section.…”

Identification of a specific region of low molecular weight phospholipases A2 (residues 21-40) as a potential target for structure-based design of inhibitors of these enzymes.

“…In addition, preincubating the enzyme with deoxycholate micelles before adding anti-PLA2 IgG did not affect the PLA2-inhibitory activity of the antibody (data not shown). This rules out an interaction of the antibody with the positively charged side chains in the C-terminal region which are of paramount importance for the binding of PLA2 to negatively charged interfaces (27). When affinity-purified IgG (1 ml of a 160 nM solution) was preadsorbed at 4°C overnight with porcine pancreatic PLA2 (12.5 ug) bound to a PVDF membrane, the PLA2 inhibitory activity of the IgG was completely abolished (data not shown).…”

“…The region of phospholipase A2 (residues [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] recognized by this antibody includes a highly conserved sequence which contains several functionally important residues of both group I and group U phospholipases A2. These data suggest that amino acid residues in this region of porcine pancreatic phospholipase A2 are accessible for interaction with inhibitors such as neutalizing antibodies and that agents specifically interacting with this region may have potent phospholipase A2 inhibitory activity.…”