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izvor podataka: crosbi

Acetylcholinesterase active centre and gorge conformations analysed by combinatorial mutations and enantiomeric phosphonates (CROSBI ID 100204)

Prilog u časopisu | izvorni znanstveni rad | međunarodna recenzija

Kovarik, Zrinka ; Radić, Zoran ; Berman, A. Harvey ; Simeon-Rudolf, Vera ; Reiner, Elsa ; Taylor, Palmer Acetylcholinesterase active centre and gorge conformations analysed by combinatorial mutations and enantiomeric phosphonates // Biochemical journal (London. 1984), 373 (2003), 33-40-x

Podaci o odgovornosti

Kovarik, Zrinka ; Radić, Zoran ; Berman, A. Harvey ; Simeon-Rudolf, Vera ; Reiner, Elsa ; Taylor, Palmer

engleski

Acetylcholinesterase active centre and gorge conformations analysed by combinatorial mutations and enantiomeric phosphonates

A series of eight double and triple mutants of mouse acetylcholinesterase (AChE ; 3.1.1.7) with substitutions corresponding to residues largely found within the butyrylcholinesterase active centre gorge (BChE ; 3.1.1.8), was analyzed to compare steady-state kinetic constants for substrate turnover and inhibition parameters for enantiomeric methylphosphonate esters. The mutations combined substitutions in the acyl pocket (F295L and F297I) with the choline binding site (Y337A and F338A) and with a side chain (E202Q) N-terminal to the active site serine, S203. The mutations affected catalysis by increasing Km and decreasing kcat, but these constants were typically affected by an order of magnitude or less, a relatively small change compared to the catalytic potential of AChE. To analyse the constraints on stereoselective phosphonylation, the mutant enzymes were reacted with a congeneric series of SP- and RP-methylphosphonates of known absolute stereochemistry. Where possible, the overall reaction rates were deconstructed into the primary constants for formation of the reversible complex and intrinsic phosphonylation. The multiple mutations greatly reduced the reaction rates of the more reactive SP-methylphosphonates, while the rates of reaction with the RP-methylphosphonates were markedly enhanced. With the phosphonates of larger steric bulk the enhancement of rates for the RP enantiomers, coupled with the reduction of the SP enantiomers, was sufficient to invert markedly the enantiomeric preference. The sequence of mutations to enlarge the size of AChE active centre gorge resembling in part the more spacious gorge of BChE did not show an ordered conversion to BChE reactivity as anticipated for a rigid template. Rather the individual aromatic residues may mutually interact to confer a distinctive stereospecificity pattern towards organophosphates.

acetylcholinesterase mutations; butyrylcholinesterase mutations organophosphate inhibition; stereoselectivity

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Podaci o izdanju

373

2003.

33-40-x

objavljeno

0264-6021

Povezanost rada

Kemija

Indeksiranost