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: Km = 0.22 +/- 0.03 mM, kcat = 128.0 +/- 17.8 s-1 and kcat/Km = 5.8 +/- 1.6 x 10(5) M-1 s-1. The L-stereoisomer of methionine competitively inhibited the enzyme reaction with a Ki 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.
Giardina
T
T
Laboratoire de Biochimie et Biologie de la Nutrition, UPRES A 6033, Faculté des Sciences et Techniques Saint-Jérôme, Marseille, France.
Biagini
A
A
Dalle Ore
F
F
Ferre
E
E
Reynier
M
M
Puigserver
A
A
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004795:Enzyme Stability; D005260:Female; D007413:Intestinal Mucosa / Q000201:enzymology*; D007526:Isoelectric Point; D007700:Kinetics; D011817:Rabbits; D013347:Subcellular Fractions; D013379:Substrate Specificity; D013552:Swine
D000581:Amidohydrolases; C019672:aminoacylase I