Determination of the active‐site serine of 6‐aminohexanoate‐dimer hydrolase

Abstract

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. Copyright © 1989, Wiley Blackwell. All rights reserved