Thomas B. Stanley

The propeptides of the vitamin K-dependent proteins possess different affinities for the vitamin K-dependent carboxylase.

The vitamin K-dependent γ-glutamyl carboxylase catalyzes the modification of specific glutamates in a number of proteins required for blood coagulation and associated with bone and calcium homeostasis. All known vitamin K-dependent proteins possess a conserved eighteen-amino acid propeptide sequence that is the primary binding site for the carboxylase. We compared the relative affinities of synthetic propeptides of nine human vitamin K-dependent proteins by determining the inhibition constants (K i ) toward a factor IX propeptide/γ-carboxyglutamic acid domain substrate. TheK i values for six of the propeptides (factor X, matrix Gla protein, factor VII, factor IX, PRGP1, and protein S) were between 2–35 nm, with the factor X propeptide having the tightest affinity. In contrast, the inhibition constants for the propeptides of prothrombin and protein C are ∼100-fold weaker than the factor X propeptide. The propeptide of bone Gla protein demonstrates severely impaired carboxylase binding with an inhibition constant of at least 200,000-fold weaker than the factor X propeptide. This study demonstrates that the affinities of the propeptides of the vitamin K-dependent proteins vary over a considerable range; this may have important physiological consequences in the levels of vitamin K-dependent proteins and the biochemical mechanism by which these substrates are modified by the carboxylase.

Identification of a vitamin K-dependent carboxylase in the venom duct of a Conus snail

Peptides from the venom ducts of cone snails (genus Conus) contain γ‐carboxyglutamate residues. The γ‐glutamyl carboxylase responsible for this post‐translational modification is localized in the microsomal fraction, strictly dependent on vitamin K, activated by ammonium sulfate, and is associated with endogenous substrate. The K m of the enzyme for vitamin K is comparable to that for the bovine carboxylase. However, a propeptide containing substrate related to the blood coagulation protein factor IX, a highly efficient substrate for the bovine enzyme, was poorly carboxylated by the Conus enzyme, suggesting differences in γ‐carboxylase recognition signal sequences and/or structural requirements at the carboxylation site.

Identification and Characterization of 4-Chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide (GSK3787), a Selective and Irreversible Peroxisome Proliferator-Activated Receptor δ (PPARδ) Antagonist

4-Chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide 3 (GSK3787) was identified as a potent and selective ligand for PPARdelta with good pharmacokinetic properties. A detailed binding study using mass spectral analysis confirmed covalent binding to Cys249 within the PPARdelta binding pocket. Gene expression studies showed that pyridylsulfone 3 antagonized the transcriptional activity of PPARdelta and inhibited basal CPT1a gene transcription. Compound 3 is a PPARdelta antagonist with utility as a tool to elucidate PPARdelta cell biology and pharmacology.

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.