Hosea N A, Berman H A, Taylor P
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
Biochemistry. 1995 Sep 12;34(36):11528-36. doi: 10.1021/bi00036a028.
We have examined the specificity of planar carboxyl and tetrahedral phosphonyl esters for mouse cholinesterases and have delineated the orientation of these ligands in the enzyme active center. The approach involved altering acyl pocket dimensions by site-specific mutagenesis of two phenylalanines and varying ligand size and enantiomer presentation. Substrate catalysis rates by wild type acetylcholinesterase (AChE) of acetyl-, butyryl-, and benzoylthiocholine diminished with increasing size of the acyl moiety. In contrast, substitution of the acyl pocket phenylalanines giving the mutants F295L and F297I of AChE yielded more efficient catalysis of the larger substrates and a specificity approaching that of butyrylcholinesterase. Extension from planar substrates to enantiomerically pure organophosphonates allowed for an analysis of enantiomeric selectivity. We found that AChE reactions are 200-fold faster with the Sp than the Rp enantiomer of of cycloheptyl methylphosphonyl thiocholine. Upon the acyl pocket size being enlarged, the Rp enantiomer became more reactive while reaction with the Sp enantiomer was slightly reduced. In fact, the F297I mutant displayed inverted stereospecificity. A visual correlation with the kinetic data has been developed by docking the ligands in the active site. Upon placement of the phosphonyl oxygen in the oxyanion hole and the leaving group being directed out of the gorge, the Rp, but not the Sp, enantiomer engendered steric hindrance between the alkoxyl group and the acyl pocket. Replacing F297 with Ile accommodated the bulky alkoxyl group of the Rp isomer in the acyl pocket, allowing similar orientations of the phosphonyl oxygen and the leaving group to the Sp isomer.(ABSTRACT TRUNCATED AT 250 WORDS)
我们研究了平面羧基酯和四面体膦酸酯对小鼠胆碱酯酶的特异性，并确定了这些配体在酶活性中心的取向。该方法包括通过对两个苯丙氨酸进行位点特异性诱变来改变酰基口袋的尺寸，以及改变配体大小和对映体呈现方式。野生型乙酰胆碱酯酶（AChE）对乙酰硫代胆碱、丁酰硫代胆碱和苯甲酰硫代胆碱的底物催化速率随着酰基部分尺寸的增加而降低。相比之下，对AChE的酰基口袋苯丙氨酸进行取代，得到突变体F295L和F297I，它们对较大底物的催化效率更高，特异性接近丁酰胆碱酯酶。从平面底物扩展到对映体纯的有机膦酸酯，可以分析对映体选择性。我们发现，AChE与环庚基甲基膦酰硫代胆碱的Sp对映体反应速度比对映体Rp快200倍。随着酰基口袋尺寸增大，Rp对映体反应性增强，而与Sp对映体的反应略有降低。事实上，F297I突变体表现出相反的立体特异性。通过将配体对接在活性位点，已建立了与动力学数据的直观关联。当膦酰氧位于氧负离子孔中且离去基团指向峡谷外时，Rp对映体（而非Sp对映体）在烷氧基和酰基口袋之间产生空间位阻。用异亮氨酸取代F297可使Rp异构体的庞大烷氧基容纳在酰基口袋中，使膦酰氧和离去基团的取向与Sp异构体相似。（摘要截短于250字）
