Crystal Structures of Brain Group-VIII Phospholipase A2 in Nonaged Complexes with the Organophosphorus Nerve Agents Soman and Sarin,

摘要

Insecticide and nerve agent organophosphorus (OP) compounds are potent inhibitors of thenserine hydrolase superfamily of enzymes. Nerve agents, such as sarin, soman, tabun, and VX exert theirntoxicity by inhibiting human acetycholinesterase at nerve synapses. Following the initial phosphonylationnof the active site serine, the enzyme may reactivate spontaneously or through reaction with an appropriatennucleophilic oxime. Alternatively, the enzyme-nerve agent complex can undergo a secondary process,ncalled “aging”, which dealkylates the nerve agent adduct and results in a product that is highly resistantnto reactivation by any known means. Here we report the structures of paraoxon, soman, and sarin complexesnof group-VIII phospholipase A2 from bovine brain. In each case, the crystal structures indicate a nonagednadduct; a stereoselective preference for binding of the PSCS isomer of soman and the PS isomer of sarinnwas also noted. The stability of the nonaged complexes was corroborated by trypsin digest and electrospraynionization mass spectrometry, which indicates nonaged complexes are formed with diisopropylfluorophosphate,nsoman, and sarin. The PS stereoselectivity for reaction with sarin was confirmed by reactionnof racemic sarin, followed by gas chromatography/mass spectrometry using a chiral column to separatenand quantitate each stereoisomer. The PS stereoisomers of soman and sarin are known to be the morentoxic stereoisomers, as they react preferentially to inhibit human acetylcholinesterase. The results obtainednfor nonaged complexes of group-VIII phospholipase A2 are compared to those obtained for other serinenhydrolases and discussed to partly explain determinants of OP aging. Furthermore, structural insights cannnow be exploited to engineer variant versions of this enzyme with enhanced nerve agent binding andnhydrolysis functions.