INACTIVATION OF PANCREATIC LIPASES BY AMPHIPHILIC REAGENTS 5-(DODECYLDITHIO)-2-NITROBENZOIC ACID AND TETRAHYDROLIPSTATIN - DEPENDENCE UPON PARTITIONING BETWEEN MICELLAR AND OIL PHASES

We have reported previously that Cys103 (SH(II)) of human pancreatic l ipase (HPL), unlike the nonessential Cys181 (SH(I)), was buried and in accessible to classical water-soluble sulfhydryl reagents. The lipolyt ic activity of HPL was lost after the labeling of the above two SH gro ups with the amphiphilic sulfhydryl reagent, 5-(dodecyldithio)-2-nitro benzoic acid (C12-TNB), suggesting that the SH(II) residue may play an important role in the hydrolytic process [Gargouri, Y., Cudrey, C., M edjoub, H., & Verger, R. (1992) Eur. J. Biochem. 204, 1063-1067]. For the present experiments, we selected dog pancreatic lipase (DPL), puri fying it for the first time, and recombinant guinea pig pancreatic lip ase (r-GPL), which both contain a buried SH(II) group but no accessibl e SH(I) group. The single SH(II) of DPL and r-GPL reacted only with th e amphiphilic SH reagent (C12-TNB), and its labeling was correlated wi th a rapid lipase inactivation. Although it is spatially remote from t he catalytic triad, the SH(II) group of pancreatic lipases, when chemi cally labeled, was found to be responsible for the loss of their lipol ytic activity. The presence of a bulky dodecyl chain, linked by a disu lfide bond to the SH(II), may have prevented the critical beta-5 loop (residues 76-85) movement by steric hindrance and consequently disturb ed the formation of the oxyanion hole. Thus, pancreatic lipase inactiv ation by the amphiphilic sulfhydryl reagent can be said to be due to t he prevention of a productive induced fit. Tetrahydrolipstatin (THL) i s an amphiphilic inactivator reacting with the essential serine of the lipase active site. Comparisons of the partitioning between the micel lar and oil phases of THL and C12-TNB were made in order to estimate t he hydrophobic-lipophilic balance of each inactivator. Its preferentia l micellar partitioning makes C12-TNB inefficient in the presence of N aTDC, whereas THL is mostly associated with the triglyceride phase eve n in the presence of bile salts. The latter physicochemical property i s probably a requirement for prototypic lipase inactivators to be effe ctive under physiological conditions, i.e., in the presence of bile an d lipids.