METAL-ION DEPENDENCE OF OLIGOSACCHARYL TRANSFERASE - IMPLICATIONS FORCATALYSIS

Oligosaccharyl transferase activity exhibits an absolute requirement f or certain divalent metal cations. Studies with reconstituted enzyme s uggest a preference for metal ions that can adopt an octahedral coordi nation geometry. In order to gain insight into the specific role of th e metal cation in catalysis, we have investigated the influence of the metal cofactor on catalytic turnover of the tripeptide substrate Bz-A sn-Leu-Thr-NHMe (1) and a closely related sulfur-containing analog, Bz -Asn(gamma S)-Leu-Thr-NHMe (2). The metal ion substitution studies rev eal that 1 is effectively turned over in the presence of several metal ions (Mn2+, Fe2+, Mg2+, and Ca2+). In contrast, 2 is only glycosylate d in the presence of the thiophilic metal cations manganese and iron. When the enzyme is reconstituted with the oxophilic cations magnesium and calcium, 2 shows minimal substrate behavior. With the amide substr ate 1, the distinct preference for manganese over magnesium may argue against direct coordination of the metal to the lipid-linked substrate pyrophosphate moiety. This fact, together with the comparative studie s with asparagine- and thioasparagine-containing tripeptides, implicat es the metal cofactor in a role that places it proximal to the peptide binding site.