Determination of the active-site serine of 6-aminohexanoate-dimer hydrolase.
Diisopropylfluorophosphate, an inhibitor of serine proteinase, was used to label 6-aminohexanoate-dimer hydrolase, a nylon oligomer degradative enzyme of Flavobacterium sp. K172. 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.
Negoro
S
S
Department of Fermentation Technology, Osaka University, Japan.
Mitamura
T
T
Oka
K
K
Kanagawa
K
K
Okada
H
H
D000345:Affinity Labels; D000581:Amidohydrolases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics; D007531:Isoflurophate / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D015202:Protein Engineering; D012694:Serine / Q000032:analysis* / Q000235:genetics; D014357:Trypsin
D000345:Affinity Labels; D010446:Peptide Fragments; D007531:Isoflurophate; D012694:Serine; D014357:Trypsin; D000581:Amidohydrolases; C019683:6-aminohexanoate-cyclic-dimer hydrolase