确定二硫键分配人力维生素K-dependent gamma-glutamyl羧化酶matrix-assisted激光解吸/电离飞行时间质谱分析。
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领带JK, Mutucumarana VP,直DL,卡里克KL,教皇RM,斯塔福德郡DW
确定二硫键分配人力维生素K-dependent gamma-glutamyl羧化酶matrix-assisted激光解吸/电离飞行时间质谱分析。
生物化学杂志。2003年11月14日,278 (46):45468 - 75。Epub 2003年9月8日。
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12963724 (在PubMed]
- 文摘
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维生素K-dependent gamma-glutamyl羧化酶是一个758氨基酸整体膜糖蛋白催化某些蛋白质的翻译后转换gamma-carboxyglutamate谷氨酸残基。羧化酶有十个半胱氨酸残基,但是他们的形式(巯基或二硫)在很大程度上是未知的。在Pudota Pudota等人,b . N。,宫城县,M。,,k W。西,k。Crabb, j·W。Misono, k . S。,Berkner k . l . (2000) Proc。国家的。学会科学。美国97年,13033 - 13038年报告说,半胱氨酸- 99和半胱氨酸- 450是羧化酶活性位点残基。我们确定所有半胱氨酸羧化酶的形式使用凝固态蛋白酶消化和matrix-assisted激光解吸/电离质谱。 The spectrum of non-reduced, trypsin-digested carboxylase revealed a peak at m/z 1991.9. Only this peak disappeared in the spectrum of the reduced sample. This peak's m/z is consistent with the mass of peptide 92-100 (Cys-99) disulfide-linked with peptide 446-453 (Cys-450). To confirm its identity, the m/z 1991.9 peak was isolated by a timed ion selector as the precursor ion for further MS analysis. The fragmentation pattern exhibited two groups of triplet ions characteristic of the symmetric and asymmetric cleavage of disulfide-linked tryptic peptides containing Cys-99 and Cys-450. Mutation of either Cys-99 or Cys-450 caused loss of enzymatic activity. We created a carboxylase variant with both C598A and C700A, leaving Cys-450 as the only remaining cysteine residue in the 60-kDa fragment created by limited trypsin digestion. Analysis of this fully active mutant enzyme showed a 30- and the 60-kDa fragment were joined under non-reducing conditions, thus confirming Cys-450 participates in a disulfide bond. Our results indicate that Cys-99 and Cys-450 form the only disulfide bond in carboxylase.