Binding of the Factor IX γ-Carboxyglutamic Acid Domain to the Vitamin K-dependent γ-Glutamyl Carboxylase Active Site Induces an Allosteric Effect That May Ensure Processive Carboxylation and Regulate the Release of Carboxylated Product

Pen-Jen Lin,David L Straight,Darrel W Stafford

doi:10.1074/jbc.m312239200

Pen-Jen Lin, David L Straight + Show 1 more

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https://doi.org/10.1074/jbc.m312239200

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Abstract

Propeptides of the vitamin K-dependent proteins bind to an exosite on gamma-glutamyl carboxylase; while they are bound, multiple glutamic acids in the gamma-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41). In addition, we characterized the effect of the Gla domain and carboxylation on propeptide and substrate binding. For the propeptide of factor IX (proFIX18), FIXproGla41, and carboxylated FIXproGla41, the Kd values were 50, 2.5, and 19.7 nM and the koff values were 273 x 10(-5), 9 x 10(-5), and 37 x 10(-5) s(-1), respectively. The koff of proFIX18 is reduced 3-fold by FLEEL and 9-fold by the Gla domain (residues 1-46) of FIX. The pre-steady state rate constants for carboxylation of FIXproGla41 was 0.02 s(-1) in enzyme excess and 0.016 s(-1) in substrate excess. The steady state rate in substrate excess is 4.5 x 10(-4) s(-1). These results demonstrate the following. 1) The pre-steady state carboxylation rate constant of FIXproGla41 is significantly slower than that of FLEEL. 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.

Highlights

Previous studies indicate that all or most of the Glu residues to be carboxylated are modified during a single substrate binding event [9, 10]
1) The pre-steady state carboxylation rate constant of FIXproGla41 is significantly slower than that of FLEEL. 2) The Gla domain plays an allosteric role in substrate-enzyme interactions
This hypothesis was based upon our measurements of the koff values of propeptides [23] from the carboxylase and on our rationale that the rate of carboxylation of a propeptide-containing substrate, FIXproGla41, would be at least as fast, and probably faster, than that of FLEEL because we expected the concentration of Glu in a bound substrate would be much higher than the non-tethered FLEEL

Summary

Introduction

Previous studies indicate that all or most of the Glu residues to be carboxylated are modified during a single substrate binding event [9, 10]. Individuals with mutations in the FIX propeptide (A–10T, A–10V, and N–9K) were identified because warfarin therapy affected the activity of factor IX much more than expected [16, 17] These mutations reduce the affinity of the propeptide for the carboxylase 10 – 600-fold. This prediction was realized as decreasing the affinity of the propeptide from ϳ2.6 to ϳ277 nM resulted in the percentage of fully carboxylated factor X increasing from about 32% to about 85% [18] All these examples can be explained by a balance between the rate of carboxylation and the rate of product dissociation, i.e. either reducing the rate of carboxylation or increasing the rate of dissociation would lead to increased amounts of undercarboxylated clotting proteins. Furie et al [21], report that mutation of either of the equivalent cysteines in the Gla domain of prothrombin does not affect carboxylation

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