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

Fibroblast activation protein alpha (FAPalpha) 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. FAPalpha exhibits a dipeptidyl peptidase IV (DPPIV)-like fold, featuring an alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain. Known DPPIV dipeptides are cleaved by FAPalpha with an approximately 100-fold decrease in catalytic efficiency compared with DPPIV. Moreover, FAPalpha, but not DPPIV, possesses endopeptidase activity toward N-terminal benzyloxycarbonyl (Z)-blocked peptides. Comparison of the crystal structures of FAPalpha and DPPIV revealed one major difference in the vicinity of the Glu motif (Glu(203)-Glu(204) for FAPalpha; Glu(205)-Glu(206) for DPPIV) within the active site of the enzyme. Ala(657) in FAPalpha, instead of Asp(663) as in DP-PIV, reduces the acidity in this pocket, and this change could explain the lower affinity for N-terminal amines by FAPalpha. This hypothesis was tested by kinetic analysis of the mutant FAPalpha/A657D, which shows on average an approximately 60-fold increase in the catalytic efficiency, as measured by k(cat)/K(m), for the cleavage of dipeptide substrates. Furthermore, the catalytic efficiency of the mutant is reduced by approximately 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 FAPalpha.

Crystal structure of human dipeptidyl peptidase IV in complex with a decapeptide reveals details on substrate specificity and tetrahedral intermediate formation.

Dipeptidyl peptidase IV (DPPIV) is a member of the prolyl oligopeptidase family of serine proteases. DPPIV removes dipeptides from the N terminus of substrates, including many chemokines, neuropeptides, and peptide hormones. Specific inhibition of DPPIV is being investigated in human trials for the treatment of type II diabetes. To understand better the molecular determinants that underlie enzyme catalysis and substrate specificity, we report the crystal structures of DPPIV in the free form and in complex with the first 10 residues of the physiological substrate, Neuropeptide Y (residues 1-10; tNPY). The crystal structure of the free form of the enzyme reveals two potential channels through which substrates could access the active site-a so-called propeller opening, and side opening. The crystal structure of the DPPIV/tNPY complex suggests that bioactive peptides utilize the side opening unique to DPPIV to access the active site. Other structural features in the active site such as the presence of a Glu motif, a well-defined hydrophobic S1 subsite, and minimal long-range interactions explain the substrate recognition and binding properties of DPPIV. Moreover, in the DPPIV/tNPY complex structure, the peptide is not cleaved but trapped in a tetrahedral intermediate that occurs during catalysis. Conformational changes of S630 and H740 between DPPIV in its free form and in complex with tNPY were observed and contribute to the stabilization of the tetrahedral intermediate. Our results facilitate the design of potent, selective small molecule inhibitors of DPPIV that may yield compounds for the development of novel drugs to treat type II diabetes.