Portal de Periódicos da CAPES

On the sulfhydryl groups of porcine pancreatic lipase and their possible role in the activity of the enzyme

1971; Elsevier BV; Volume: 242; Issue: 3 Linguagem: Inglês

10.1016/0005-2744(71)90151-3

1878-1454

R. Verger, Louis Sarda, P. Desnuelle,

Hormonal and reproductive studies

Porcine pancreatic lipase contains two SH groups (SH1 and SH11). The SH1 group is reactive in the native enzyme towards sulfhydryl reagents such as 5,5′-dithiobis(2-nitrobenzoic) acid, p-chloromercuribenzoate and N-ethylmaleimide, but not towards iodoacetate and iodoacetamide. The SH11 group is unreactive except towards phenylmercuric ions. Both groups are reactive towards all reagents in the presence of 0.3% sodium dodecyl sulfate or 8 M urea. These observations led to the preparation of two well-defined S-substituted lipase derivatives: mono 5-thio-2-nitrobenzoic acid (TNB) lipase in which the SH1 group was selectively blocked by a TNB radical and diphenyl mercuric (DPM) lipase in which the SH1 and SH11 groups were both blocked by a phenylmercuric radical. A “mixed” derivative containing a TNB radical on the SH1 group and a phenylmercuric radical on the SHII group could also be prepared by incubation of mono TNB-lipase with phenylmercuric chloride. All these reactions were found to be fully reversed by incubation with an excess of thiol. An intramolecular thiol-disulfide reaction was observed to occur when mono TNB-lipase was treated with 0.3% sodium dodecyl sulfate or 8 M urea or, although more slowly, when a solution of lipase in these reagents was oxidized by air. This reaction resulted in the formation of an additional disulfide bridge in the lipase molecule. The kinetic parameters Km and vmax of the hydrolysis of emulsified triglycerides by mono TNB-lipase, DPM-lipase and native lipase were determined and compared. Substitution of the SH1 group by a TNB radical induced a 10-fold increase in Km whereas vmax was unaffected. Substitution of both the SH1 and SH11 groups by a phenylmercuric radical induced a 40% decrease in vmax while Km was unchanged. Lipase regenerated by thiols from the derivatives had exactly the same parameters as the native enzyme. A possible interpretation of these data is that the SH1 group is at or near the site responsible for the attachment of lipase at hydrophobic interfaces whereas the SH11 group is near the catalytic site. The properties of both groups suggest that they are situated in an hydrophobic region of the molecule.