Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 402, Issue 2, Pages 281-288Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S0003-9861(02)00087-5
Keywords
phosphonate monoester; covalent reactivity; serine proteinase; catalytic antibody
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Funding
- NCI NIH HHS [CA80312] Funding Source: Medline
- NHLBI NIH HHS [HL59746, HL44126] Funding Source: Medline
- NIAID NIH HHS [AI31268, AI46029] Funding Source: Medline
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Phosphonate monoesters have been assumed to serve as noncovalent transition state analogs for enzymes capable of catalyzing transacylation reactions. Here, we present evidence for the covalent reaction of certain serine proteinases and peptidase antibody fragments with monophenyl amino(4-amidinophenyl)methanephosphonate derivatives. Stable adducts of the N-biotinylated monophenyl ester with trypsin and antibody fragments were evident under conditions that disrupt noncovalent interactions. The reaction was inhibited by the active-site-directed reagent diisopropyl fluorophosphate. Mass spectrometry of the fragments from monoester-labeled trypsin indicated phosphonylation of the active site. Irreversible inhibition of trypsin- and thrombin-catalyzed hydrolysis of model substrates was observed. Kinetic analysis of inactivation of trypsin by the N-benzyloxycarbonylated monoester suggested that the first-order rate constant for formation of covalent monoester adducts is comparable to that of the diester adducts (0.47 vs 2.0 min(-1)). These observations suggest that the covalent reactivity of phosphonate monoesters contributes to their interactions with serine proteinases, including certain proteolytic antibodies. (C) 2002 Elsevier Science (USA). All rights reserved.
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