SEMISYNTHETIC GLYCOPROTEINS - PREPARATION OF GLYCOSYLATED RIBONUCLEASE S' ANALOGS

Glycopeptide analogues of ribonuclease S-peptide, des 16-20 [(Gal beta )Thr3]-S-peptide and des 16-20 [(Gal beta)Thr6]-S-peptide, were synthe sized by the solid-phase procedure based on the Fmoc chemistry. Reasso ciation of the synthetic glycopeptides with S-protein yielded enzymati cally active, glycosylated RNase S' variants. The conformational prope rties of the S-peptide analogues in aqueous buffer and in the presence of trifluoroethanol were investigated by circular dichroism spectrosc opy. The activation curves of the S-protein with varying amounts of th e synthetic analogues, using C > p as the substrate, were determined a nd the dissociation constants (K(d)), the free energies of binding (-D ELTAG), and the kinetic parameters (K(m) and k2) for the RNase S' addu cts were calculated and compared with the corresponding values obtaine d for the S-peptide. The properties of the semisynthetic, glycosylated enzymes agree with the results of previous investigations on the conf ormational and catalytic features of S-peptide analogues and related R Nase S'.