Mechanism of Interaction of Esterases with Organophosphates
The Ministry of Science, Education and Sports of the Republic of Croatia (2002-2006)
Principal Investigator: Vera Simeon
Since January 1 2005, Principal Investigator Zrinka Kovarik
This project deals with interactions of esterases and organophosphates (triesters of phosphorus acids), and other inhibitors and substrates. Some nerve warfare agents and pesticides are organophosphates (OP). Three esterases will be studied: acetylcholinesterase (AChE, EC 184.108.40.206), butyrylcholinesterase (BChE, EC 220.127.116.11) and a phosphoric triester hydrolase, paraoxonase (EC 18.104.22.168).
Organophosphates inhibit serine esterases, AChE and BChE, but some of these compounds are substrates of paraoxonase. AChE has a vital function to terminate cholinergic neurotransmission by rapid hydrolysis of acetylcholine. Phosphorylation of the active site of this enzyme is the main cause of OP toxicity.
BChE is also phosphorylated by OP but has no obvious physiological function. Therefore this enzyme acts as a scavenger of OP and of some other esters (pesticides and drugs). Phosphoric triester hydrolases break down OP to non-toxic products thus playing an important role in detoxication. Our studies are focused on stereospecificity of AChE and BchE, and on the role of amino acid residues in their active site gorge.
The source of enzymes will be DNA-derived enzymes, native and purified preparations. The project comprises interactions with substrates, symmetric and chiral inhibitors: organophosphates, esters of carbamic acids, phenotiazine and quinuclidine derivatives. Kinetic constants of interactions of the enzymes with substrates, inhibitors and reactivators will be determined. Several kinetic models will be evaluated on obtained experimental data. Standardisation of the enzyme activity measurements and determination of phenotypes of BChE and paraoxonase in human blood are parts of the programme. Conventional (spectrophotometry and pH-stat titration) and stopped-flow techniques will be used for measuring the enzyme activities.