Identification of the catalytic residues of carboxylesterase from Arthrobacter globiformis by diisopropyl fluorophosphate-labeling and site-directed mutagenesis.
The role of amino acid residues in the enzymatic activity of carboxylesterase from Arthrobacter globiformis was analyzed by diisopropyl fluorophosphate (DFP) labeling and site-directed mutagenesis. The electrospray ionization mass spectrometric (ESI-MS) analysis of the esterase, covalently labeled by DFP, showed stoichiometric incorporation of the inhibitor into the enzyme. The further comparison of endopeptidase-digested fragments between native and DFP-labeled esterase by fast atom bombardment mass spectrometric (FAB-MS) analysis as well as site-directed mutagenesis indicated that Ser59 in the consensus sequence Ser-X-X-Lys, which is conserved exclusively in penicillin-binding proteins and some esterases, served as a catalytic nucleophile. In addition, the results obtained from analysis of the mutants at position 62 suggested the importance of the basic amino acid side chain at this position, and suggested the significance of this residue acting directly as a general base rather than its involvement in the maintenance of the optimum hydrogen-bonding network at the active site.
Genomic Science Laboratories, Dainippon Sumitomo Pharma Co., Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan. masako-nishizawa@ds-pharma.co.jp
Masako
M
Nishizawa
Yoshiyasu
Y
Yabusaki
Masaharu
M
Kanaoka
D001173:Arthrobacter / Q000201:enzymology*; D055162:Biocatalysis; D043182:Carboxylesterase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006868:Hydrolysis; D007531:Isoflurophate / Q000378:metabolism*; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011722:Pyrethrins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013194:Staining and Labeling; D013237:Stereoisomerism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011722:Pyrethrins; D007531:Isoflurophate; C007051:chrysanthemic acid; D043182:Carboxylesterase