Solution structure of porcine pancreatic phospholipase A2.

Abstract

The lipolytic enzyme phospholipase A2 (PLA2) is involved in the degradation of high‐molecular weight phospholipid aggregates in vivo. The enzyme has very high catalytic activities on aggregated substrates compared with monomeric substrates, a phenomenon called interfacial activation. Crystal structures of PLA2s in the absence and presence of inhibitors are identical, from which it has been concluded that enzymatic conformational changes do not play a role in the mechanism of interfacial activation. The high‐resolution NMR structure of porcine pancreatic PLA2 free in solution was determined with heteronuclear multidimensional NMR methodology using doubly labeled 13C, 15N‐labeled protein. The solution structure of PLA2 shows important deviations from the crystal structure. In the NMR structure the Ala1 alpha‐amino group is disordered and the hydrogen bonding network involving the N‐terminus and the active site is incomplete. The disorder observed for the N‐terminal region of PLA2 in the solution structure could be related to the low activity of the enzyme towards monomeric substrates. The NMR structure of PLA2 suggests, in contrast to the crystallographic work, that conformational changes do play a role in the interfacial activation of this enzyme.

A Novel Prokaryotic Phospholipase A2

“…A nitrogen atom of His 64 , which may act as a general base-catalyst, is 5.5 Å apart from the calcium(II) ion. Similar alignments of imidazole and carboxylate groups are well known in the active center of the other calcium-dependent PLA 2 s, such as porcine pancreatic PLA 2 (12).…”

“…Several structural studies of PLA 2 in solution (12) and in crystal (13)(14)(15)(16)(17)(18)(19)(20) have been reported. The crystal structural studies of secreted PLA 2 s have shown that the overall structures of class I PLA 2 s are similar to those of class II enzymes.…”

The Crystal Structure of Prokaryotic Phospholipase A2

“…The formation of a hydrogenbonding network extended from the catalytic site is incomplete when compared with the crystal structure (11). However, the solution structure of the porcine PLA 2 complexed with a competitive inhibitor and micelles showed that the N-terminal region and surface loop take stable conformations and form the complete hydrogen-bonding network, as seen in the crystal structure (12).…”

“…However, the catalytic hydrogen-bonding network and the substrate-binding site are flexible in the calcium-bound form of the bacterial PLA 2 . Interestingly, recent studies of eukaryotic PLA 2 s using the NMR method (11)(12)(13) showed the flexible feature of the enzyme at a part of catalytic hydrogen-bonding network and the substrate-binding site. The flexibility in the crystal structure of the calcium-bound S. violaceoruber PLA 2 may be associated with these observations in the solution structures of eukaryotic PLA 2 s.…”

“…As a striking difference, eukaryotic PLA 2 s have 6 -8 disulfide bonds, whereas the bacterial enzyme has only two disulfide bonds. Over 150 primary structures of PLA 2 have been already determined, and the crystal (4 -10) and NMR structures (11)(12)(13) of several PLA 2 s, such as bovine and porcine pancreatic PLA 2 s, snake and bee venom PLA 2 s, and human synovial fluid PLA 2 , have been solved.…”