Mutation of tryptophan residues in lipoprotein lipase. Effects on stability, immunoreactivity, and catalytic properties.

Previous studies had pointed to an important function of a putative exposed loop in the C-terminal domain of lipoprotein lipase for activity against emulsified lipid substrates. This loop contains 3 tryptophan residues (Trp390, Trp393, and Trp394). We have expressed and characterized lipase mutants with tryptophan to alanine substitutions at positions 55, 114, 382, 390, 393, and 394 and a double mutant at residues 393 and 394. The substitutions in the N-terminal domain (W55A and W114A) led to poor expression of completely inactive lipase variants. Heparin-Sepharose chromatography showed that mutant W114A eluted at the same salt concentration as inactive wild-type monomers, indicating that this substitution prevented subunit interaction or led to an unstable dimer. In contrast, all mutants in the C-terminal domain were expressed as mixtures of monomers and dimers similarly to the wild-type. The dimers displayed at least some catalytic activity and had the same apparent heparin affinity as the active wild-type dimers. The mutants W390A, W393A, W394A, and W393A/W394A had decreased reactivity with the monoclonal antibody 5D2, indicating that the 5D2 epitope is longer than was reported earlier, or that conformational changes affecting the epitope had occurred. The mutants W390A, W393A, W394A, and W393A/W394A had decreased catalytic activity against a synthetic lipid emulsion of long-chain triacylglycerols (IntralipidR) and in particular against rat lymph chylomicrons. The most pronounced decrease of activity was found for the double mutant W393A/W394A which retained only 6% of the activity of the wild-type lipase, while 70% of the activity against water-soluble tributyrylglycerol was retained. In the case of chylomicrons also the affinity for the substrate particles was lowered, as indicated by severalfold higher apparent Km values. This effect was less prominent with the synthetic lipid emulsion. We conclude that the tryptophan cluster Trp390-Trp393-Trp394 contributes to binding of lipoprotein lipase to lipid/water interfaces. Utilizing different lipid substrates in different physical states, we have demonstrated that the tryptophan residues in the C-terminal domain may have a role also in the productive orientation of the enzyme at the lipid/water interface.