A novel fluorescence assay to study propeptide interaction with gamma-glutamyl carboxylase

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttransl ational modification of select glutamate residues of its vitamin K-dependen t substrates to gamma-carboxyglutamate. In this report, we describe a new f luorescence assay that is sensitive and specific for the propeptide binding site of active carboxylase. We employed the assay to make three important observations: (1) A tight binding fluorescein-labeled consensus propeptide can be used to quantify the active fraction of the enzyme. (2) The off-rate for a fluorescein-labeled factor IX propeptide was 3000-fold slower than t he rate of carboxylation, a difference that may explain how carboxylase can carry out multiple carboxylations of a substrate during the same binding e vent. (3) We show evidence that substrate binding to the active site modifi es the propeptide binding site of carboxylase. The significant (9-fold) dif ferences in off-rates for the propeptide in the presence and absence of its co-substrates may represent a release mechanism for macromolecular substra tes from the enzyme. Additionally, sedimentation velocity and equilibrium e xperiments indicate a monomeric association of enzyme with propeptide. Furt hermore, the carboxylase preparation is monodisperse in the buffer used for our studies.