A MOLECULAR BIOLOGY-BASED APPROACH TO RESOLVE THE SUBUNIT ORIENTATIONOF LIPOPROTEIN-LIPASE

The subunit orientation of a dimeric enzyme influences the mechanism o f action and function, To determine the subunit arrangement of lipopro tein lipase (LPL), a molecular biology-based approach was initiated, A n eight amino acid linker region was engineered between two LPL monome rs and expressed in COS-7 cells, The resultant tandem-repeat molecule (LPLTR) was lipolytically active and had kinetic parameters, salt inhi bition, cofactor-dependent activity, heparin-binding characteristics, and a functional unit size very similar to the expressed native human enzyme, By these criteria, LPLTR was the functional equivalent of nati ve LPL. Considering the length of the linker peptide (no more than 24 Angstrom), monomers in the tethered molecule were restricted to a head -to-tail subunit arrangement, Since LPLTR demonstrated native enzyme-l ike properties while constrained to this subunit arrangement, these re sults provide the first compelling evidence that native LPL monomers a re arranged in a head-to-tail subunit orientation within the active di mer, Thus, LPL function in physiology, lipolysis, and binding to cell- surface components must now be addressed with this subunit orientation in mind, The utility of the tandem-repeat approach to resolve the sub unit arrangement of an obligate dimer has been demonstrated with LPL a nd could be generalized for use with other oligomeric enzymes.