PROLINE-RICH DOMAIN AND GLYCOSYLATION ARE NOT ESSENTIAL FOR THE ENZYMATIC-ACTIVITY OF BILE SALT-ACTIVATED LIPASE - KINETIC-STUDIES OF T-BAL, A TRUNCATED FORM OF THE ENZYME, EXPRESSED IN ESCHERICHIA-COLI

We have expressed and purified a truncated recombinant human milk bile salt-activated lipase (T-BAL) from the T7 expression system in Escherichia coli. This T-BAL contains the N-terminal 538 residues of the 722-residue native enzyme. The purified T-BAL, when assayed with PANA (p-nitrophenyl acetate), had a specific activity of 64 +/- 2 units/mg (n = 4), as compared to 52 units/mg for the native enzyme. Because the recombinant T-BAL expressed in E. coli is not glycosylated, these results indicated that the highly glycosylated C-terminal region of BAL is not essential for catalytic function. Heat inactivation patterns of native BAL and T-BAL were found to be similar, further suggesting that the folding of T-BAL is similar to that of the catalytic domain of the native enzyme. With the availability of a sufficient amount of recombinant T-BAL, the specificity and kinetics of T-BAL and native BAL were compared. Fluorescence studies of T-BAL indicated that it has a slightly higher affinity for the monomeric form of taurocholate with a dissociation constant (K-A) of 0.32 mM, compared with the reported 0.37 mM for the native enzyme. Further kinetic analysis indicated that there are enzyme specificity changes revealed with the use of PANA and PANB (p-nitrophenyl butyrate) as substrates. When assayed in the presence of taurocholate, T-BAL has a higher turnover rate constant with p-nitrophenyl acetate with the p-nitrophenyl butyrate, which was found to be in contrast to native BAL. However, similar to the native enzyme, T-BAL still has a higher substrate specificity constant with PANB than with PANA, because of the much lower Michaelis-Menten constant with PANB. The essential requirement of bile salt micelles as fatty acid acceptor in the BAL catalysis was also similar between T-BAL and native BAL. However, T-BAL was more resistant to the inactivation effect of a high concentration of taurocholate.