Benton M E, Price P A, Suttie J W
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706-1569, USA.
Biochemistry. 1995 Jul 25;34(29):9541-51. doi: 10.1021/bi00029a031.
The vitamin K-dependent carboxylase processes multiple glutamic acid residues to gamma-carboxyglutamic acid (Gla) residues in a limited number of proteins. The targeted proteins are synthesized with an amino-terminal propeptide which has been shown to play an important role in gamma-carboxylation. The specificity of the enzyme for each potential Gla site, the direction of carboxylation, and the influence of a bound propeptide on these events are not understood. Des-gamma-carboxy forms of bone Gla protein (BGP), which contain potential Gla residues at positions 17, 21, and 24, were employed as model substrates to determine the multi-site-specificity of the enzyme. Recombinant bovine des-gamma-carboxylated proBGP (rdproBGP) and heat-decarboxylated BGP (dBGP), lacking a propeptide, were used as substrates for a bovine liver carboxylase, and the in vitro reaction products were analyzed for the formation of 14CO2 Gla. The di-Gla species was found to be the predominant product of in vitro carboxylation of both rdproBGP and dBGP at less than saturating concentrations of each substrate. Carboxylation of both substrates occurred preferentially at the more C-terminal potential Gla sites, residues 21 and 24. A similar pattern of carboxylation was observed with a rat bone cell carboxylase, suggesting no species or tissue variation in the enzyme specificity. Some tricarboxylated product accumulated during carboxylation of rdproBGP but not dBGP, suggesting that the covalently bound propeptide directs more complete carboxylation of the Gla domain. In addition, monocarboxylated rdproBGP was found to accumulate in the absence but not in the presence of a free noncovalently attached propeptide, indicating that free propeptide affects more efficient carboxylation of rdproBGP.
维生素K依赖性羧化酶可将多种谷氨酸残基加工成有限数量蛋白质中的γ-羧基谷氨酸（Gla）残基。靶向蛋白在合成时带有一个氨基末端前肽，该前肽已被证明在γ-羧化过程中起重要作用。目前尚不清楚该酶对每个潜在Gla位点的特异性、羧化方向以及结合的前肽对这些过程的影响。骨Gla蛋白（BGP）的去γ-羧基形式在第17、21和24位含有潜在的Gla残基，被用作模型底物来确定该酶的多位点特异性。缺乏前肽的重组牛去γ-羧基化前BGP（rdproBGP）和热脱羧BGP（dBGP）被用作牛肝羧化酶的底物，并对体外反应产物进行14CO2 Gla形成的分析。在每种底物浓度未饱和时，发现二Gla物种是rdproBGP和dBGP体外羧化的主要产物。两种底物的羧化优先发生在更靠近C末端的潜在Gla位点，即第21和24位残基。用大鼠骨细胞羧化酶观察到类似的羧化模式，表明该酶的特异性不存在物种或组织差异。在rdproBGP羧化过程中积累了一些三羧化产物，但dBGP没有，这表明共价结合的前肽可引导Gla结构域更完全的羧化。此外，发现单羧化的rdproBGP在没有游离非共价连接的前肽时会积累，而在有游离前肽时则不会积累，这表明游离前肽会影响rdproBGP更有效的羧化。
