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 ex posed loop in the C-terminal domain of lipoprotein lipase for activity against emulsified lipid substrates. This loop contains 3 tryptophan residues (Trp(390), Trp(393), and Trp(394)). We have expressed and cha racterized lipase mutants with tryptophan to alanine substitutions at positions 55, 114, 382, 390, 393, and 394 and a double mutant at resid ues 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 th e same salt concentration as inactive wild-type monomers, indicating t hat this substitution prevented subunit interaction or led to an unsta ble dimer. In contrast, all mutants in the C-terminal domain were expr essed as mixtures of monomers and dimers similarly to the wild-type. T he 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 th an 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 o f long-chain triacylglycerols (Intralipid(R)) and in particular agains t 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 w ater-soluble tributyrylglycerol was retained, In the case of chylomicr ons also the affinity for the substrate particles was lowered, as indi cated by severalfold higher apparent K-m values. This effect was less prominent with the synthetic lipid emulsion. We conclude that the tryp tophan cluster Trp(390) Trp(393)-Trp(394) contributes to binding of li poprotein 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.