Activation Mechanisms for Venom Phospholipase A2 Enzymes

[摘要] Acyl imidazolide activation of venom phospholipase A2 enzymes was studied by a variety of kinetic and chemical means in order to determine the nature of the reaction between the protein and the activator and to identify the amino-acid residues involved. The conductimetric methods of Lawrence, (1971) were extensively modified and improved. The proposal of Drainas, (1978) that activation by acylation was complementary to activation by calcium was studied in detail. Nitrogen-based chelators were used to show that the metal ion dependence was dominated by the inhibitory effects of transition metal ion impurities, with copper and zinc being the most powerful inhibitors. A kinetic investigation showed that these metal ions were not simple competitive inhibitors for calcium. The kinetic results showed that long-chain fatty acylation had no effect on the metal-ion dependence. The importance of using transition metal ion chelators (eg o-phenanthroline) in such kinetic studies was demonstrated and this study revealed that the affinity constant for calcium was ca 5muM (ie 50 fold less that previously stated). A new polyacrylamide gel method was developed to study small basic peptides; this had a lower detection limit at octapeptides, but could routinely detect decapeptides using the Coomassie blue detection method. This was used to monitor purification of bee venom components and to characterise peptide fragments of bee venom phospholipase A2. Short chain acyl imidazolides were found to block the activation site without giving full activation. This was consistent with models in which a long hydrophobic chain was required either to act as a substrate penetration probe or else to change the conformation of the enzyme. The conformation change model was supported by further studies of the resistance of the activated enzyme to a variety of inhibitory reagents. Use of radiolabelled acyl imidazolide activators showed that the acyl residue did not remain bound to the activated enzyme under acidic denaturing conditions. Cyanogen bromide fragments and fragments obtained by peptidase treatment retained insignificant amounts of the labelled acyl residue. Group modifying agents were tested as blockers or modifiers of the activation reaction. The results eliminated any direct involvement of the N-terminal residue or of lysine side chains. Modification of tyrosine and arginine residues changed the nature of activation but did not affect reaction with the activators. Demonstration that the enzyme could be activated by p-bromophenacyl bromide in the presence, but not in the absence of long-chain fatty acid pointed to the direct involvement of an active histidine residue. Comparison of the sequences of activatable and non-activatable enzymes indicated that a histidine residue at 20-26 might be important.

[发布机构] University:University of Glasgow

[关键词] Molecular biology