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 ...
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