The hog intestinal mucosa acylase I: Subcellular localization, isolation, kinetic studies and biological function

The soluble acylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) from hog intestinal mucosa was 11 000-fold purified for the first time using a new four-step procedure involving an immunoaffinity chromatography. The resulting protein, which had an isoelectric point of 5.2 and a M-r of 90 000 was composed of two apparently identical N-acylated polypeptide chains. Its amino acid composition was comparable to that of hog kidney acylase I. The enzyme had a pH optimum at 8.0 and required Zn2+ or Co2+. The optimal temperature for the acylase reaction was 40 degrees C and the activation energy of thermodenaturation was estimated at 260 kJ mol(-1). The enzyme was strongly inhibited when preincubated with chelating agents, by diethyl pyrocarbonate under histidine-modifying conditions as well as by sulfhydryl compounds. The reaction of the purified enzyme with the synthetic substrate furylacryloyl-L-methionine was partly characterized as follows: K-m = 0.22 +/- 0.03 mM, k(cat) = 128.0 +/- 17.8 s(-1) and kc(at)/K-m = 5.8 +/- 1.6 x 10(5) M-1 s(-1). The L-stereoisomer of methionine competitively inhibited the enzyme reaction with a K-i of 3.4 +/- 0.2 mM. It is suggested that acylase I might not only be involved in the catabolism of intracellular N-acylated protein but also be responsible for the biological utilization of N-acylated food proteins.