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
D. Downs et al., 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, Biochemistry, 33(26), 1994, pp. 7979-7985
We have expressed and purified a truncated recombinant human milk bile
salt-activated lipase (T-BAL) from the T7 expression system in Escher
ichia 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 re
combinant T-BAL expressed in E. coli is not glycosylated, these result
s indicated that the highly glycosylated C-terminal region of BAL is n
ot essential for catalytic function. Heat inactivation patterns of nat
ive BAL and T-BAL were found to be similar, further suggesting that th
e folding of T-BAL is similar to that of the catalytic domain of the n
ative enzyme. With the availability of a sufficient amount of recombin
ant T-BAL, the specificity and kinetics of T-BAL and native BAL were c
ompared. Fluorescence studies of T-BAL indicated that it has a slightl
y higher affinity for the monomeric form of taurocholate with a dissoc
iation constant (K-A) of 0.32 mM, compared with the reported 0.37 mM f
or the native enzyme. Further kinetic analysis indicated that there ar
e 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-nitroph
enyl 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. T
he essential requirement of bile salt micelles as fatty acid acceptor
in the BAL catalysis was also similar between T-BAL and native BAL. Ho
wever, T-BAL was more resistant to the inactivation effect of a high c
oncentration of taurocholate.