Propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state

Certain individuals with combined deficiencies of vitamin K-dependent proteins have a mutation, L394R, in their ␥-glutamyl carboxylase causing impaired glutamate binding. The sequence surrounding Leu 394 is similar in all known carboxylases, suggesting that the region is functionally important. To test this hypothesis we made the following mutant enzymes: W390A, Y395A, S398A, W399A, and H404A. We purified the enzymes and corrected the activity measurements for active enzyme concentration. Carboxylases W390A, S398A, and H404A had activities similar to that of wild type; however, Y395A and W399A had lower activities than did wild type. In the following descriptions we include our previously reported results for L394R. Kinetic studies with the substrate FLEEL, revealed K m values of 0.5 (wild type), 6.5 (L394R), 15 (Y395A), and 24 (W399A) mM. The k cat values relative to wild type were 51% (L394R), 1% (Y395A), and 2% (W399A). The k cat /K m values were 24-fold (L394R) and >2000-fold lower for Y395A and W399A than for wild-type carboxylase. Inhibition of FLEEL carboxylation by the competitive inhibitor, Boc-mEEV, gave K i values of 0.013 (wild type), 1.4 (L394R), 2.1 (Y395A), and >5 (W399A) mM. The Y395A propeptide affinity was similar to that of wild type, but those of L394R and W399A were 16 -22-fold less than that of wild type. Results of kinetic studies with a propeptide-containing substrate were consistent with results of propeptide binding and FLEEL kinetics. Although propeptide and vitamin K binding in some mutants were affected, our data provide compelling evidence that glutamate recognition is the primary function of the conserved region around Leu 394 .

Section: Vitamin K-dependent Carboxylase Glutamate Binding Residuesmentioning

confidence: 52%

A Conserved Region of Human Vitamin K-dependent Carboxylase between Residues 393 and 404 Is Important for Its Interaction with the Glutamate Substrate

Mutucumarana

Acher

Straight

et al. 2003

“…Sugiura et al (10) showed that carboxylase does not catalyze epoxidation of KH 2 in the absence of propeptide or FLEEL. The reaction is fastest, ϳ10-fold faster than with any single component, with both FLEEL and propeptide.…”

Section: Discussionmentioning

confidence: 99%

Effect of Vitamin K-dependent Protein Precursor Propeptide, Vitamin K Hydroquinone, and Glutamate Substrate Binding on the Structure and Function of γ-Glutamyl Carboxylase

Mutucumarana

Lin

et al. 2010

The ␥-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 NaHCO 3 , 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.The vitamin K-dependent ␥-glutamyl carboxylase is an endoplasmic reticulum integral membrane enzyme. It post-translationally modifies certain proteins important in several physiologically important areas, including blood coagulation. The modification involves the addition of a carboxyl group to the ␥-carbon of glutamic acid residues in the amino portion of the vitamin K-dependent substrate.In addition to the glutamic acid and CO 2 , which provides the added carboxyl group, other substrates required for the reaction are vitamin K hydroquinone (KH 2 ) 4 and oxygen. During carboxylation KH 2 is converted to the epoxide; thus carboxylase is also an epoxidase.The primary interaction between carboxylase and most of its protein substrates is through the propeptides of the substrates. Binding is of high affinity, which allows the substrate to bind long enough for multiple carboxylations to occur.In addition to being the primary binding site for carboxylase, the propeptide also exerts an effect on carboxylase catalysis. Factors IX and X and prothrombin propeptides, when bound to carboxylase, increase the activity of the enzyme toward small glutamate-containing substrates (1-5).For the most part these results suggest that the propeptides induce a conformational change or stabilize a more active conformation in the carboxylase active site. The efficiency of the change appears to be similar for the propeptides of prothrombin and factors IX and X. The fact that these propeptides have different sequences implies that ...

“…7, a and b). The cells contain endogenous carboxylase, whose ability to recycle KH 2 to KO was unknown because the carboxylase requires activation by VKD proteins (29,30), which were present in only trace amounts in these cells (31). Therefore, the experiment was repeated in cells pretreated with carboxylase siRNA prior to the addition of KO.…”

Section: Vkorc1 Reduces Ko To Kh 2 To Drivementioning

confidence: 99%

The Vitamin K Oxidoreductase Is a Multimer That Efficiently Reduces Vitamin K Epoxide to Hydroquinone to Allow Vitamin K-dependent Protein Carboxylation

Rishavy

Hallgren

Wilson

et al. 2013

Background: How the vitamin K oxidoreductase (VKORC1) supports vitamin K-dependent protein carboxylation is poorly understood. Results: VKORC1 multimers efficiently perform both reactions that reduce vitamin K, and the inactive monomer in wild type mutant heteromers suppresses reduction. Conclusion: VKORC1 fully reduces vitamin K required for carboxylation. Significance: Multimers are important in VKORC1 mechanism and wild type mutant heteromers impact patients with warfarin resistance.