Exploring the Active Center of Human Acetylcholinesterase with Stereomers of an Organophosphorus Inhibitor with Two Chiral Centers

详细信息    查看全文

• 作者：Arie Ordentlich ; Dov Barak ; Chanoch Kronman ; Hendrik P. Benschop ; Leo P. A. De Jong ; Naomi Ariel ; Ruth Barak ; Yoffi Segall ; Baruch Velan ; and Avigdor Shafferman
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：March 9, 1999
• 年：1999
• 卷：38
• 期：10
• 页码：3055 - 3066
• 全文大小：173K
• 年卷期：v.38,no.10(March 9, 1999)
• ISSN：1520-4995

文摘

The stereoselectivity of the phosphonylation reaction and the effects of adduct configurationon the aging process were examined for human acetylcholinesterase (HuAChE) and its selected activecenter mutants, using the four stereomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman).The reactivity of wild type HuAChE toward the P_S-soman diastereomers was 4.0-7.5 × 10⁴-fold higherthan that toward the P_R-diastereomers. Aging of the P_SC_S-somanyl-HuAChE conjugate was also >1.6 ×10⁴-fold faster than that of the corresponding P_RC_S-somanyl adduct, as shown by both reactivation andelectrospray mass spectrometry (ESI/MS) experiments. On the other hand, both processes exhibited verylimited sensitivity to the chirality of the alkoxy group C_{ages/gifchars/alpha.gif" BORDER=0>} of either P_S- or P_R-diastereomers. Thesestereoselectivities presumably reflect the relative participation of the enzyme in stabilization of the Michaeliscomplexes and in dealkylation of the respective covalent conjugates, and therefore could be utilized forfurther probing of the HuAChE active center functional architecture. Reactivities of HuAChE enzymescarrying replacements at the acyl pocket (F295A, F297A, and F295L/F297V) indicate that stereoselectivitywith respect to the soman phosphorus chirality depends on the structure of this binding subsite, but thisstereoselectivity cannot be explained only by limitation in the capacity to accommodate the P_R-diastereomers. In addition, these acyl pocket enzyme mutants display some (5-10-fold) preference forthe P_RC_R-soman over the P_RC_S-stereomer, while reactivity of the hydrophobic pocket mutant enzymeW86F toward the P_RC_S-soman resembles that of the wild type HuAChE. Residue substitutions in theH-bond network (E202Q, E450A, Y133F, and Y133A) and the hydrophobic pocket (F338A, W86A, W86F,and Y337A) result in a limited stereoselectivity for the P_SC_S- over the P_SC_R-stereomer. Aging of theP_S-somanyl conjugates with all the HuAChE mutant enzymes tested practically lacked stereoselectivitywith respect to the C_{ages/gifchars/alpha.gif" BORDER=0>} of the alkoxy moiety. Thus, the inherent asymmetry of the active center does notseem to affect the rate-determining step of the dealkylation process, possibly because both the P_SC_S- andthe P_SC_R-somanyl moieties yield the same carbocationic intermediate.