Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2010 Oct 8;285(41):31502-8. doi: 10.1074/jbc.M110.143297. Epub 2010 Aug 17.
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 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.
γ-谷氨酰羧化酶利用四种底物,催化维生素 K 依赖性蛋白中某些谷氨酸残基的羧化作用。酶如何将底物聚集在一起以促进催化是理解该酶结构和功能的一个重要问题。前肽是维生素 K 依赖性蛋白与羧化酶的主要结合位点。它也是羧化酶活性的效应物。我们检验了这样一个假设,即底物的结合会引起羧化酶的变化,并进一步引起底物-酶相互作用的变化。此外,我们还研究了前肽序列如何影响底物-酶相互作用。为了研究这些问题,我们使用荧光标记的前肽来测量与羧化酶的亲和力。我们还测量了几种前肽增加羧化酶催化活性的能力。最后,我们确定了底物:维生素 K 氢醌、五肽 FLEEL 和 NaHCO(3)对前肽-羧化酶复合物稳定性的影响。我们发现前肽与羧化酶的亲和力有很大差异。相比之下,测试的前肽对羧化酶的催化活性有相似的影响。FLEEL 和维生素 K 氢醌都能稳定前肽-羧化酶复合物。两者一起的效果比单独使用任何一种都要好。我们的结论是,前肽和底物对羧化酶的影响控制着底物结合的顺序,以确保有效的、特异性的羧化作用。