LIPOAMIDASE ACTIVITY IN NORMAL AND MUTAGENIZED PANCREATIC CHOLESTEROLESTERASE (BILE SALT-STIMULATED LIPASE)

Purified human milk lipoamidase was digested with endoproteinase Lys-C and the digested peptides were subjected to gas-phase microsequence a nalysis. The sequencing of three isolated peptides of human milk lipoa midase revealed the identity of this protein with human milk bile salt -stimulated lipase (pancreatic cholesterol esterase). The identity of the cholesterol esterase with lipoamidase was confirmed by expressing a recombinant form of rat pancreatic cholesterol esterase and testing for lipoamidase activity of the recombinant protein. The results showe d that the recombinant cholesterol esterase displayed both lipolytic a nd lipoamidase activities and was capable of hydrolysing triacetin and lipoyl-4-aminobenzoate (LPAB). The mechanisms of the esterase and ami dase activities of the enzyme were further tested by determining enzym e activity in a mutagenized cholesterol esterase with a HiS435 --> Gln 435 substitution. This mutation has been shown previously to abolish e nzyme activity against esterase substrates [DiPersio, Fontaine and Hui (1991) J. Biol. Chem. 266, 4033-40361. We showed that the mutagenized protein was effective in hydrolysing the amidase substrate LPAB and d isplayed similar enzyme kinetics to those of the native enzyme. These data indicate that the mechanism for the cholesterol esterase hydrolys is of lipoamides is different from that of the hydrolysis of substrate s with an ester linkage. The presence of an enzyme in the gastrointest inal tract capable of both ester and amide hydrolysis suggests an impo rtant role for this protein in the digestion and absorption processes.