We have reported previously that Cys103 (SHII) of human pancreatic lipase (HPL), unlike the nonessential Cys181 (SHI), was buried and inaccessible to classical water-soluble sulfhydryl reagents. The lipolytic activity of HPL was lost after the labeling of the above two SH groups with the amphiphilic sulfhydryl reagent, 5-(dodecyldithio)-2-nitrobenzoic acid (C12-TNB), suggesting that the SHII residue may play an important role in the hydrolytic process [Gargouri, Y., Cudrey, C., Medjoub, H., & Verger, R. (1992) Eur. J. Biochem. 204, 1063–1067]. For the present experiments, we selected dog pancreatic lipase (DPL), purifying it for the first time, and recombinant guinea pig pancreatic lipase (r-GPL), which both contain a buried SHII group but no accessible SHI group. The single SHII of DPL and r-GPL reacted only with the amphiphilic SH reagent (C12-TNB), and its labeling was correlated with a rapid lipase inactivation. Although it is spatially remote from the catalytic triad, the SHII group of pancreatic lipases, when chemically labeled, was found to be responsible for the loss of their lipolytic activity. The presence of a bulky dodecyl chain, linked by a disulfide bond to the SHII, may have prevented the critical β−5 loop (residues 76–85) movement by steric hindrance and consequently disturbed the formation of the oxyanion hole. Thus, pancreatic lipase inactivation by the amphiphilic sulfhydryl reagent can be said to be due to the prevention of a productive induced fit. Tetrahydrolipstatin (THL) is an amphiphilic inactivator reacting with the essential serine of the lipase active site. Comparisons of the partitioning between the micellar and oil phases of THL and C12-TNB were made in order to estimate the hydrophobic-lipophilic balance of each inactivator. Its preferential micellar partitioning makes C12-TNB inefficient in the presence of NaTDC, whereas THL is mostly associated with the triglyceride phase even in the presence of bile salts. The latter physicochemical property is probably a requirement for prototypic lipase inactivators to be effective under physiological conditions, i.e., in the presence of bile and lipids. © 1993, American Chemical Society. All rights reserved.
