BENZYLOXYCARBONYLPROLYLPROLINAL, A TRANSITION-STATE ANALOG FOR PROLYLOLIGOPEPTIDASE, FORMS A TETRAHEDRAL ADDUCT WITH CATALYTIC SERINE, NOTA REACTIVE CYSTEINE

N-Benzyloxycarbonyl-L-prolyl-L-[1-C-13]prolinal was synthesized starti ng with reduction of L-[1-C-13]Pro to L-[1-C-13]prolinol, followed by coupling with N-benzyloxycarbonyl-L-Pro to N-benzyloxycarbonyl-L-Pro-L -[1 -C-13]prolinol (Z-Pro-[1-C-13]prolinol), and finally oxidation of the alcohol to the aldehyde with dimethyl sulphoxide. While the C-13 N MR chemical shift of the aldehyde carbon is 202 p.p.m., that of the al dehyde hydrate is between 91.6 and 91.8 p.p.m., that of the dithiothre itol adduct is between 74.8 and 75.0 p.p.m., and that in the presence of the serine protease prolyl oligopeptidase is at 92.3 p.p.m.. The li newidth of the latter is 114 Hz, roughly consistent with the molecular mass of 80 kDa reported for the enzyme, Inverse detection experiments gave a H-1 resonance at 5.29 p.p.m. with a linewidth of 80 Hz, also c onsistent with the expected chemical shift and linewidth for a hemiace tal bound to such a large enzyme, while the free hydrate gave resonanc es at 5.18 and 5.25 p.p.m., with very much narrower linewidths. It is concluded that Z-Pro-prolinal, a putative transition-state analogue fo r prolyl oligopeptidase, forms a tetrahedral complex with the enzyme a t its catalytic serine, rather than at a neighbouring cysteine that wa s found to be highly reactive according to chemical modification studi es.