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Use of VX and V‐analog compounds to probe the spatial requirements of the active site of wild type and mutant human paraoxonase 1
Author(s) -
Otto Tamara Caviston,
Yeung David T,
Cerasoli Douglas M,
Lenz David E
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1008.11
Subject(s) - chemistry , leaving group , isopropyl , stereochemistry , hydrolysis , catalysis , mutant , alkyl , paraoxonase , active site , medicinal chemistry , enzyme , biochemistry , organic chemistry , gene
Human paraoxonase 1 (HuPON1) exhibits catalytic activity toward the organophosphorus nerve agent VX, but the catalytic mechanism mediating this activity is unresolved. To examine the effect of substrate volume on activity, the hydrolysis of five analogs of VX was examined. The analogs were designed such that the O‐ ethyl alkyl group of VX was replaced with O‐propyl (V1), O‐isopropyl (V2) or O‐isobutyl (V3) on the retained group, or the 2‐diisopropyl alkyl groups were replaced with 2‐diethyl (V4) or 2‐dimethyl (V5) on the leaving group. Both K m and k cat values for V1, V2, and V3 decreased as the retained group increased in size. In contrast, reducing the size of the leaving group (V4 and V5) had little effect on K m and progressively decreased k cat . The H1 15W mutant of HuPON1 hydrolyzed V1, V4 and V5, whereas no turnover of V2 or V3 was detected. Further analyses determined that V3 was recognized as a competitive inhibitor of VX hydrolysis by the H115W mutant. The results indicate that the volume of both the retained and leaving groups can affect binding of these substrates to HuPON1 and also suggest that the R‐chain of the retained group may directly interact with residue 115. Together, the results allow an estimate of the size of the HuPON1 binding pocket to be made. This work was supported by NIH Centers of Excellence Grant U‐54 NS 058183‐01 Center for Catalytic Bioscavenger Medical Defense Research. TCO was a NRC research fellow.