The endo-β-glucuronidase heparanase mediates mammalian heparan sulfate catabolism, and is of considerable medical interest due to its prominent role in cancer aggression and metastasis. Biochemical studies of heparanase are currently hampered by a lack of suitable chromogenic or fluorogenic assay substrates, instead relying on lengthy multistep procedures to measure activity. Herein, we demonstrate that N’,6-O’-bis-sulfated 4-methylumbelliferyl heparan sulfate disaccharide is a competent fluorogenic heparanase substrate. Despite somewhat slow turnover, the high sensitivity of 4-methylumbelliferyl fluorescence provides a wide signal window that enables both enzyme turnover and inhibition kinetics measurements. Crystal structures with heparanase also provide the first ever observation of a substrate in an activated 1S3 conformation, highlighting previously unknown interactions involved in turnover. Our results pave the way for the design of further improved HPSE substrates that may enable rapid assessment of enzyme activity, which in turn will drive development of new heparanase inhibitors for therapeutic use.
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