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 (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 beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)