期刊
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
卷 1698, 期 1, 页码 27-36出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2003.10.002
关键词
fluoroacetate dehalogenase; L-2-haloacid dehalogenase; asparagine residue; deamidation; site-directed mutagenesis; mass spectrometry
Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon-fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the a-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp 105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the L-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation. (C) 2003 Elsevier B.V. All rights reserved.
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