Evidence that the zymogen of phospholipase A2 binds to a negatively charged lipid-water interface

Abstract

Evidence is presented that the zymogen of porcine pancreatic phospholipase A~ (prophospholipase A2) interacts with a lipid-water interface provided that the interface has a net negative surface charge. Fluorescence spectroscopy and non-equilibrium gel filtration indicate that binding of prophospholipase A 2 (proPLA) to mixed detergent micelles is dependent on the presence of an anionic detergent. Prophospholipase binding is accompanied by a change in the environment of the single tryptophan residue qualitatively similar to that observed when the active enzyme, phospholipase A s (PLA), binds to micelles. In addition, the rate of tryptic activation of prophospholipase is significantly reduced in the presence of negatively-charged mixed micelles, whereas no change in rate occurs when neutral micelles are present. These observations suggest that the lack of catalytic activity of the zymogen toward organized substrates carrying a negative surface charge cannot be explained by a failure to bind at the lipidwater interface.

The Basis for k*cat Impairment in Prophospholipase A2 from the Anion-Assisted Dimer Structure,

“…It should be noted that the change in the intensity in the difference spectrum reflects the fraction of the enzyme in the E*−MJ33 form, while the shape of the difference spectra, in particular, peaks at 288 and 292 nm, is attributed to the Trp3 environment. These difference spectra for PLA2 or proPLA2 binding MJ33 at the interface are strikingly similar to those observed by the Utrecht group ( , ) with the enzyme binding to SDS. The intensity change at 292 nm as a function of the mole fraction of the inhibitor () suggests that the affinities of MJ33 for PLA2 and proPLA2 are not significantly different.…”

Optically detected magnetic resonance studies of porcine pancreatic phospholipase A2 binding to a negatively charged substrate analog

“…It is of interest that when proPA2 was titrated with either single-chain or double-chain anionic substrate analogues, only a relatively small perturbation of the Trp environment was observed from the phosphorescence spectra and the ODMR transition frequencies. This agrees with the results obtained by Hille et al (1983a) andVolwerk et al (1984); the zymogen of porcine PA2 interacts with anionic or negatively charged mixed detergent micelles, but the extent of the perturbation of the single Trp environment is less than that found for the active enzyme.…”

“…In these "pseudomicellar" complexes only PA2 but not the zymogen becomes superactivated. A related observation is that of Volwerk et al (1984), who found that titration of PA2 with SDS-containing C16-PN micelles leads to a large (ca. 200%) increase in fluorescence, whereas only a relatively small (less than 50%) fluorescence enhancement is found by using proPA2.…”

“…The zymogen of pancreatic PA2 does not bind to organized, zwitterionic lipid-water interfaces, probably because of its distorted lipid binding domain (Volwerk & de Haas, 1982), although it interacts with interfaces having a net negative surface charge (Hille et al, 1983a;Volwerk et al, 1984). It was, therefore, of interest to study the interaction of this protein with both double-chain and single-chain anionic substrate analogues.…”

Rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex. A steady-state and time-resolved anisotropy study

“…If the decrease in dynamic freedom found for Trp-3 also occurs for other residues, the decrease in configurational entropy for ProPLA2 upon binding may be of sufficient magnitude to prevent complex formation. Recent studies indicate that ProPLA2 can bind to negatively charged micelles (Volwerk et al, 1985). In this case, the electrostatic interaction of the negatively charged lipids with positively charged residues on the surface of ProPLA2 may compensate for any loss of configurational entropy that binding entails.…”