Naslov
Uloga pojedinih domena aktivnog mjesta acetilkolinesteraze pri vezanju organofosfata i oksima
(The role of acetylcholinesterase active site domains in binding organophosphates and oximes)

Autori
Kovarik, Zrinka

Vrsta, podvrsta i kategorija rada
Ocjenski radovi, doktorska disertacija

Fakultet
Prirodoslovno matematički fakultet, Kemijski odsjek

Mjesto
Zagreb

Datum
27.03

Godina
2002

Stranica
XIV, 85

Mentor
Simeon, Vera i Weygand-Đurašević, Ivana

Ključne riječi
Acetilkolinesteraza ; Butirilkolinesteraza ; Organofosforni spojevi ; Stereoselektivnost ; Oksimi ; Inhibicija ; Reaktivacija
(Acetylcholinesterase ; Butyrylcholinesterase ; Organophosphorus compounds ; Stereoselectivity ; Oximes ; Inhibition ; Reactivation)

Sažetak
Organophosphates are potent inhibitors of acetylcholinesterase (AChE ; EC 3.1.1.7) and butyrylcholinesterase (BChE ; EC 3.1.1.8) by reacting covalently with active centre serine. In attempting to determine the amino acid residues within the active site gorge involved in the interaction with organophosphorus compounds and pyridinium oximes, three double and four triple AChE mutants were prepared with combined substitutions in the acyl pocket (F295L, F297I), choline binding site (Y337A, F338A) and a residue, neighbouring the catalytic serine, E202Q. The rate constants of inhibition of the mouse AChE, its mutants and mouse BChE by SP- and RP-cycloheptyl- (CHMP), isopropyl- (iPrMP), and 3, 3-dimethylbutyl- (DMBMP) methylphosphonyl thiocholine enantiomers, and O, O-dimethyl O-(2, 2-dichlorovinyl) phosphate (DDVP) were determined. The phosphorylated enzymes were subjected to reactivation by the pyridinium oximes, HI-6 (1-(2’ -hydroxyiminomethyl-1'-pyridinium)-3-(4''-carbamoyl-1''-pyridinium)-2-oxapropane dichloride) and 2-PAM (2-(hydroxyiminomethyl)-1-methylpyridinium iodide) and the relevant kinetic constants were determined. The SP enantiomers of the methylphosphonate esters are more reactive in forming the conjugate with AChE and mutants than RP enantiomers. Opening of the choline binding site by double mutations Y337A/F338A enhanced inhibition rates 2-fold for all inhibitors except for SP-CHMP thiocholine which inhibited the mutant at a similar rate as wild type AChE. Upon the F297I/Y337A mutations, RP enantiomers of CHMP and iPrMP thiocholines became more reactive than SP enantiomers while reaction with the SP enantiomers was slightly reduced, displaying inverted stereospecificity. The SP conjugates were more susceptible to HI-6 and 2-PAM reactivation than RP conjugates. HI-6 gives faster rates of reactivation of phosphorylated AChE and its mutants than 2-PAM, while BChE conjugates were more susceptible to 2-PAM reactivation. The rates for HI-6 and 2-PAM reactivation of phosphorylated wild type AChE. were the highest for the iPrMP-conjugate which present the least interference of three SP phosphonates for oxime entry to the base of the gorge and attack on phosphorus oxygen. Substitution in the choline binding site (Y337A) and acyl pocket (F295L or F297I) enhanced reactivation rates by HI-6 and 2-PAM with the highest acceleration achieved for enzyme phosphorylated by phosphonate with more bulky alkoxy moiety.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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