Benkouka_1982_Eur.J.Biochem_128_331

Following complete sequence analysis of the 449 amino acids in porcine pancreatic lipase [J. De Caro et al. (1981) Biochim. Biophys. Acta, 671, 129-138], the position of the six disulfide bridges and of the two free thiols of the protein was investigated using a variety of techniques. Three bridges (Cys-4--Cys-10, Cys-237--Cys-261 and Cys-433--Cys-449) were easily identified in the peptic digest of lipase at pH 2.0. In the latter digest, two other bridges (Cys-285--Cys-296 and Cys-299--Cys-304) were also identified by means of the cystine peptide constituted by two peptide segments: Ala281-Gly-Phe-Pro-Cys-Asp-Ser287 and Thr292-Ala-Asn-Lys-Cys-Phe-Pro-Cys-Pro-Ser-Glu-Gly-Cys-Pro-Gln-Met307. A disulfide bridge, formed by Cys-285 and one of the three half-cystines of the other segment, connected the two peptide moieties. A second disulfide bridge linked the two remaining half-cystines. It was not possible to split any peptide bond between Cys-296 and Cys-304 with proteolytic enzymes. The determination of the pairing of the four half-cystines was resolved as follows. Bond Lys-295--Cys-296 was cleaved with trypsin. A single cycle of Edman degradation was then performed on the peptide compound, thus freeing, in particular, Cys-296 of the peptide bond (296-297). Cys-296 only retained its S-S connection with the half-cystine partner. At the completion of the above operations, the two peptide segments (282-287) and (297-307) could be separated. Consequently it was concluded that Cys-285 is linked to Cys-296. Therefore Cys-299 and Cys-304 are paired. The most reactive SHI group of the enzyme was localized on Cys-181 by condensation of the native protein with radioactive N-ethylmaleimide. The alkylation of the SHII group required previous denaturation of the molecule at alkaline pH. The results obtained for the characterization of the SHII group suggested the existence of two isomeric forms, the SHII being either on Cys-101 or Cys-103 and the bridge alternately between Cys-90--Cys-103 or Cys-90--Cys-101. It is not yet known whether these two forms pre-exist in native lipase or result from an exchange reaction. The bridge Cys-90--Cys-101 was characterized in a thermolytic digest of a cyanogen bromide fragment (CN II) of the protein. However, another bridge involving Cys-181 was also found in the digest. This bridge is considered as being an artefact. It is possible that considering the treatments undergone by the large peptide (CN II), the SH groups of lipase were oxidized and transformed in an S-S bridge. The disulfide bridges of lipase form relatively small loops along the main chain. This arrangement is consistent with a high flexibility of the molecule. As reported earlier [R. Verger et. al. (1971) Biochim. Biophys. Acta. 242, 580-592], the SHI group is not essential for lipase activity. The role of the SHII group should be more precisely investigated.