Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation.
Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation.
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DOI：
10.1073/pnas.97.24.13033
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发表时间：
2000-11
影响因子：
11.1
通讯作者：
B. Pudota;M. Miyagi;K. Hallgren;K. West;J. Crabb;K. Misono;K. Berkner
中科院分区：
文献类型：
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作者：
B. Pudota;M. Miyagi;K. Hallgren;K. West;J. Crabb;K. Misono;K. Berkner
维生素K依赖性羧化酶修饰并赋予参与止血、细胞生长控制和钙稳态的活性维生素K依赖性蛋白质。使用一种新的机制，羧化酶转换维生素K氢醌（KH（2））氧化的自由能，将谷氨酸转化为碳负离子中间体，随后攻击CO（2），产生γ-羧化谷氨酸产物。羧化酶如何影响这种转化的了解很少，因为活性位点尚未确定。Dowd及其同事[Dowd，P.，赫什莱恩河哈姆，S。W. & Naganathan，S.（1995）Science 269，1684-1691]提出弱碱（半胱氨酸）产生能够产生碳负离子的强碱（含氧KH（2））。为了定义活性位点并测试该模型，我们鉴定了参与这些反应的氨基酸。N-乙基马来酰亚胺抑制环氧化和羧化反应，KH（2）预孵育对这两种活性有同等的保护作用。（14）C-N-乙基马来酰亚胺修饰的人羧化酶的氨基酸分析显示1.8-2.3个活性残基和7 × 10（8）cpm/hr/mg的比活性。胰蛋白酶消化和液相色谱电喷雾质谱鉴定Cys-99和Cys-450为活性位点残基。丝氨酸突变减少了环氧化和羧化，至0。2%（Cys-99）或1%（Cys-450），并使谷氨酰底物的K（m）s增加6至8倍。一些活性的保留表明增强半胱氨酸/丝氨酸亲核性的机制，这是许多活性位点硫醇酶共有的性质。这些研究代表了在确定羧化酶活性位点方面的突破，提出了一个修正模型，其中谷氨酰底物间接地与至少一个硫醇配位，形成催化复合物，该复合物电离硫醇以启动KH（2）氧化。
The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH(2)) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO(2), generating the gamma-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684-1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH(2)) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH(2) preincubation. Amino acid analysis of (14)C- N-ethyl maleimide-modified human carboxylase revealed 1.8-2.3 reactive residues and a specific activity of 7 x 10(8) cpm/hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0. 2% (Cys-99) or 1% (Cys-450), and increased the K(m)s for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH(2) oxygenation.