Vasantha P. Mutucumarana

Expression and Characterization of the Naturally Occurring Mutation L394R in Human γ-Glutamyl Carboxylase

Patients with mutation L394R in γ-glutamyl carboxylase have a severe bleeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially corrects this deficiency. To characterize L394R, we purified recombinant mutant L394R and wild-type carboxylase expressed in baculovirus-infected insect cells. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor IXs propeptide and vitamin KH2. Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher K i for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower V max/K m toward the substrate FLEEL in the presence of the propeptide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-fold higher K m for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher K m for vitamin KH2. The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH2 binding properties are altered in the L394R mutant. Compared with its wild-type counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide.

Characteristics of recombinant W501S mutated human γ‐glutamyl carboxylase

Summary.  A mutation (W501S) in the vitamin K‐dependent γ‐glutamyl carboxylase (VKC) that leads to a congenital bleeding disorder was recently discovered in two patients. To characterize the enzyme defect, recombinant VKC‐W501S was expressed in and purified from insect cells. The major effect of the mutation appears to be to decrease the affinity of the carboxylase for the propeptide of its substrates. This observation agrees with recent data that place part of the propeptide binding site within residues 495–513 of VKC. Additionally, we demonstrate that the affinity between descarboxy osteocalcin (d‐OC) and VKC remains unaffected by the W501S mutation. This confirms earlier data that the high‐affinity site for d‐OC is not located on the propeptide binding domain of VKC. Two properties of the enzyme suggest an explanation for the observation that vitamin K supplementation ameliorates the effects of the mutation: (i) since full carboxylation requires the propeptide to remain bound to the enzyme sufficiently long for full carboxylation, a reduced affinity can cause its premature release before carboxylation is complete; (ii) propeptide binding results in a decrease of the KM for vitamin K hydroquinone in wild‐type, but not in mutant carboxylase, resulting in increased vitamin K requirement of affected subjects.

Purification of gamma-glutamyl carboxylase from bovine liver.

Publisher Summary This chapter describes an affinity purification method specifically designed for bulk isolation of γ -glutamyl carboxylase from bovine liver. The principle of this methodology is based on two factors: (1) the affinity of γ -glutamyl carboxylase to a recombinant substrate FIXQ/S and (2) the different biophysical properties of different detergents. Starting from 8 g of microsomal proteins, a 7000-fold purification is routinely achieved with about 30% recovery, which is equivalent to 300–400 μ g of γ -glutamyl carboxylase with 80–90% purity. FIXQ/S is a recombinant peptide that contains the propeptide and Gla domain sequences of human factor IX. Because of the affinity between the propeptide and γ -glutamyl carboxylase, FIXQ/S works well as an affinity ligand in the purification. Factor IXQ/S contains two mutations in the propeptide sequences, which are introduced to improve the proteolytic stability of this peptide without changing its affinity to γ -glutamyl carboxylase. This modification increases the lifetime of the Affi-FIXQ/S column.

Effect of vitamin K-dependent protein precursor propeptide, vitamin K hydroquinone, and glutamate substrate binding on the structure and function of γ-glutamyl carboxylase

The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates causes changes to the carboxylase and in turn to the substrate-enzyme interactions. In addition we investigated how the sequences of the propeptides affected the substrate-enzyme interaction. To study these questions we employed fluorescently labeled propeptides to measure affinity for the carboxylase. We also measured the ability of several propeptides to increase carboxylase catalytic activity. Finally we determined the effect of substrates: vitamin K hydroquinone, the pentapeptide FLEEL, and NaHCO3, on the stability of the propeptide-carboxylase complexes. We found a wide variation in the propeptide affinities for carboxylase. In contrast, the propeptides tested had similar effects on carboxylase catalytic activity. FLEEL and vitamin K hydroquinone both stabilized the propeptide-carboxylase complex. The two together had a greater effect than either alone. We conclude that the effect of propeptide and substrates on carboxylase controls the order of substrate binding in such a way as to ensure efficient, specific carboxylation.

A fluorescent method to determine vitamin K-dependent gamma-glutamyl carboxylase activity

The gamma (γ)-glutamyl carboxylase is a key enzyme in vitamin K-dependent carboxylation of proteins involved in hemostasis and inflammation. It is an endoplasmic enzyme posttranslationally converting glutamic acid residues into γ-carboxyglutamic acid residues in proteins. The activity of tissue derived γ-glutamyl carboxylase is commonly assayed by incorporation of H¹⁴CO₃⁻ into synthetic peptides and subsequent quantification using liquid scintillation counting. We present a nonradioactive assay using a fluorescein isothiocyanate-labeled short peptide that can be readily detected in its unmodified and γ-glutamyl carboxylated form by reversed-phase HPLC. This method offers a convenient alternative to the established radioactive labeling techniques.