Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein α

Abstract

Fibroblast activation protein α (FAPα) is highly expressed in epithelial cancers and has been implicated in extracellular matrix remodeling, tumor growth, and metastasis. We present the first high resolution structure for the apoenzyme as well as kinetic data toward small dipeptide substrates. FAPα exhibits a dipeptidyl peptidase IV (DPPIV)-like fold, featuring an α/β-hydrolase domain and an eight-bladed β-propeller domain. Known DPPIV dipeptides are cleaved by FAPα with an ∼100-fold decrease in catalytic efficiency compared with DPPW. Moreover, FAPα, but not DPPIV, possesses endopeptidase activity toward N-terminal benzyloxycarbonyl (Z)-blocked peptides. Comparison of the crystal structures of FAPα and DPPW revealed one major difference in the vicinity of the Glu motif (Glu 203-Glu204 for FAPα; Glu205-Glu 206 for DPPIV) within the active site of the enzyme. Ala 657 in FAPα, instead of Asp663 as in DP-PIV, reduces the acidity in this pocket, and this change could explain the lower affinity for N-terminal amines by FAPα. This hypothesis was tested by kinetic analysis of the mutant FAPα/A657D, which shows on average an ∼60-fold increase in the catalytic efficiency, as measured by Kcat/K m, for the cleavage of dipeptide substrates. Furthermore, the catalytic efficiency of the mutant is reduced by ∼350-fold for cleavage of Z-Gly-Pro-7-amino-4-methylcoumarin. Our data provide a clear understanding of the molecular determinants responsible for the substrate specificity and endopeptidase activity of FAPα. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.