Proline-rich domain and glycosylation are not essential for the enzymic 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 (KA) 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 than with p-nitrophenyl butyrate, which was found to be in contrast to native BAL.(ABSTRACT TRUNCATED AT 250 WORDS)
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Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
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Tang
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Wang
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D004952:Esters / Q000378:metabolism; D005260:Female; D006031:Glycosylation; D006801:Humans; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008895:Milk, Human / Q000201:enzymology*; D008969:Molecular Sequence Data; D011392:Proline; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D002787:Sterol Esterase; D013379:Substrate Specificity; D013656:Taurocholic Acid / Q000378:metabolism
D004952:Esters; D011994:Recombinant Proteins; D013656:Taurocholic Acid; D011392:Proline; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase; D008049:Lipase