Specificity of prolyl endopeptidase

Abstract

A series of tetrapeptides, Cbz(Bz)-Gly-X-Leu-Gly, were synthesized and the kinetic parameters, k cat and k cat K m , determined for their hydrolyses by prolyl endopeptidase from Flavobacterium. The peptides with X = N-Me-Ala, Sar and Ala as well as the standard substrate (X = Pro) were found to be good substrates, while those with X = α-aminobutyryl, Hyp, Ser and Gly were poor substrates, and those with X = pipecolyl, α-aminoisobutyryl, N-Me-Val, N-Me-Leu, Hyp(O-Bzl) and Ser(O-Bzl) were not cleaved at all. These results suggest that the specificity-determining site or Sl subsite of the enzyme is designed to fit exactly the proline residue of the substrate with allowance for the residues carrying substituents at the N and/or C α which must not exceed the size of the pyrrolidine ring of proline.