Open AccessDetermination of the active‐site serine of 6‐aminohexanoate‐dimer hydrolase
Author(s) -
NEGORO Seiji,
MITAMURA Toshihide,
OKA Kaneo,
KANAGAWA Kazuo,
OKADA Hirosuke
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb15144.x
Subject(s) - enzyme , serine , biochemistry , dimer , chemistry , hydrolase , peptide , active site , amino acid , enzyme assay , serine hydrolase , site directed mutagenesis , peptide sequence , oligopeptide , oligomer , stereochemistry , microbiology and biotechnology , biology , organic chemistry , mutant , gene
Diisopropylfluorophosphate, an inhibitor of serine proteinase, was used to label 6‐aminohexanoate‐dimer hydrolase, a nylon oligomer degradative enzyme of Flavobacterium sp. KI72. More than 95% of the enzyme activity was lost upon incorporation of 1–1.5 molecules inhibitor/subunit of the enzyme. The tryptic peptide of the labeled enzyme was purified by HPLC (reverse‐phase partition) and its amino acid sequence was identified. Radioactivity was found to be incorporated into an 8‐amino‐acid peptide (108His‐Leu‐Leu‐Met‐Ser‐Val‐Ser‐Lys115). Amino acid alteration from Ser to Ala at the position 112 by site‐directed mutagenesis caused loss of enzyme activity to below the detection threshold (1% of the activity of the parental enzyme). These results indicate that Ser112 is essential for the activity.