Objetivo Phospholipase A2 is a member of an important class of lipid degrading enzymes. To design relevant mutants of possible industrial significance and inhibitors of pharmaceutical use it is necessary to understand the molecular basis of the function of the enzyme. One interesting aspect of the activity of these enzymes is much higher activity for phospholipids in aggregates such as micelles and monolayers than for single phospholipid molecules in water.The general objective of this project is to understand how phospholipase A2 binds monomeric phospholipids and develop computational tools for targeting the most effective amino acids to substitute to modify molecular properties.Simulation of a phospholipase A2 monomeric substrate complex:Molecular simulation of a modelled phospholipase, calcium and phospholipid complex in water suggested that the substrate is located by relatively few directional interactions. These are primarily the interaction of 1 phosphate oxygen and the carbonyl oxygen of the cleaved bond with the calcium ion. The role of tyrosine 69 in further orientating the substrate, by hydrogen bonding and other phosphate oxygen, is suggested by the modelling but does not persist during the 48 ps simulation. This interaction may be more important when aggregated substrate are bound to the enzyme. The water molecule that is the proposed nucleophile is present during the simulation although it approaches the calcium ion.Charge engineering:The effect of mutating an uncharged nitrogen-terminal alpha helix capping residue to a charged residue (N89D) has been investigated. The residue was not less stable than expected (the stabilities of 2 other mutants were as expected). Finite difference calculations on this mutant indicate the large, compensating differences in interaction energies that yield the relatively small overall change in stability of this mutant. Hence a small error in the individual components give a large error in the overall stability. The activity of the double mutant N89D/E92K is low and suggests that residue 92 is important in interacting with aggregated substrate either directly or possibly via calcium.A reasonable understanding of how phospholipase A2 binds monomeric substrates has been obtained. This understanding is being exploited in engineering phopholipases A2 with modified specificities. Simple electrostatic calculations have been fairly successful in predicting the change in stability of proteins due to charge engineering. Improved calculations indicate large compensating interaction energies for the mutant that did not behave as expected. The activity of a double mutant N89D/E92K is low and suggests that residue 92 is important in interacting with aggregated substrates either directly or possibly via calcium. An understanding of the dynamic behaviour of monolayers of phospholipids has also developed. The monolayers are intrinsically more flexible than bilayers. Explicit description of the water appears to be essential to describe the behaviour of amphiphile monolayers. Simulation of the monolayer-phospholipase complex is now in progress. Ámbito científico natural scienceschemical sciencesinorganic chemistryalkaline earth metalsnatural sciencesbiological sciencesbiochemistrybiomoleculeslipidsnatural scienceschemical sciencesorganic chemistryaminesnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes Programa(s) FP1-BAP - Multiannual research action programme (EEC) in the field of biotechnology (BAP), 1985-1989 Tema(s) Data not available Convocatoria de propuestas Data not available Régimen de financiación CSC - Cost-sharing contracts Coordinador BBSRC Institute of Food Research Aportación de la UE Sin datos Dirección Reading Laboratory Shinfield RG2 9AT Reading Reino Unido Ver en el mapa Coste total Sin datos
