Journal
PROCESS BIOCHEMISTRY
Volume 41, Issue 2, Pages 477-482Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2005.07.003
Keywords
choline oxidase; histidine; serine; diethylpyrocarbonate; chemical modification; phenylmethanesulfonyl fluoride
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This report concentrated on clarifying the histidine and serine roles in choline oxidase (ChOx) biocatalytic activity using chemical modifications. The serine and histidine residues on ChOx were chemically modified by diethylpyrocarbonate (DEPC) and phenylmethanesulfonyl fluoride (PMSF) at 100 mM Tris-HCl buffer, pH 8. The time course, inhibition kinetics and conformational changes of ChOx were investigated using spectrophotometric, fluorescence and Far-UV CID techniques. Enzyme activity decreased of 90% and 50% in the presence of DEPC and PMSF, respectively. The double reciprocal lineweaver-burk plots indicated a competitive and an uncompetitive inhibition pattern for DEPC and PMSF, respectively suggesting the location of histidine in the enzyme active site and that of serine in the outside of active site, but somewhere close to it. (c) 2005 Elsevier Ltd. All rights reserved.
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