Aminopeptidase p mediated detoxification of organophosphonate analogues of sarin: mechanistic and stereochemical study at the phosphorus atom of the substrate.
The activity of the aminopeptidase P from Escherichia coli in hydrolyzing a series of organophosphonate sarin analogues (1-6) was evaluated. The enzymatic rates of hydrolysis for methylphosphonate 1 with a methoxy group attached to the phosphorus center were 7- to 15-fold higher than those for the corresponding analogues 2-6. Double mutant R153W/R370L was able to hydrolyze the S(p) enantiomer of racemic 1 at a considerable rate. This mutant allowed the preparation of the R(p) isomer of the sarin analogue 1. All the mutants, R370L, R153A, W88L, R153L/R370L, and R153W/R370L, preferred the formation of (S(p))-8 to that of the corresponding (R(p))-8 enantiomer and displayed a better enantiomeric excess of products, by 1.4- to 2-fold as compared to the wild-type enzyme. Enzymatic hydrolysis of O,O-diisopropyl-p-nitrophenyl phosphate (9) in H(2) (18)O led to the formation of the (18)O-labeled O,O-diisopropyl phosphate product and confirmed that the catalytic reaction starts with cleavage of the P--O bond. From chemical and kinetic studies, the utilization of an optically pure S(p) enantiomer of O-methyl-p-nitrophenyl methylphosphonothioate (S(p))-MNMPT, 7) has demonstrated that the enzymatic reaction proceeds through a displacement mechanism and generates a chiral product in situ with an inversion of stereochemical configuration at the phosphorus atom. The results also lead to the conclusion that alteration of the active site through site-directed mutagenesis can result in a preference for (S(p))-MNMPT (7) rather than the R(p) isomer.
Huang
Li-Fang
LF
Institute of Chemistry, Academia Sinica, 128 Academia Road Sec. 2, Taipei 115, Taiwan.
Su
Benson
B
Jao
Shu-Chuan
SC
Liu
Kwang-Ting
KT
Li
Wen-Shan
WS
D000626:Aminopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010758:Phosphorus / Q000737:chemistry*; D012524:Sarin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism
D017931:DNA Primers; D009943:Organophosphorus Compounds; D010758:Phosphorus; D012524:Sarin; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase