Title : Structural and kinetic analysis of the substrate specificity of human
fibroblast activation protein alpha
Abstract :
- 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 apo enzyme 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
Output (sent_index, trigger,
protein,
sugar,
site):
Output(Part-Of) (sent_index,
protein,
site):
- 4. Known DPPIV, dipeptides
- 6. enzyme, site
- 7. FAPalpha, Ala(657)
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):