对鸟苷diphospho-D-mannose脱氢酶从铜绿假单胞菌。结构分析的蛋白水解作用有限。
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Roychoudhury年代,Chakrabarty K Ho YK Chakrabarty
对鸟苷diphospho-D-mannose脱氢酶从铜绿假单胞菌。结构分析的蛋白水解作用有限。
J生物化学杂志。1992年1月15日,267 (2):990 - 6。
- PubMed ID
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1370473 (在PubMed]
- 文摘
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藻朊酸盐被认为是一个主要的毒力因子在铜绿假单胞菌的致病性肺囊性纤维化的病人。鸟嘌呤核苷diphospho-D-mannose脱氢酶(EC 1.1.1.132 GDPmannose脱氢酶)是一种海藻酸生物合成途径中的关键酶催化氧化的鸟苷diphospho-D-mannose (GDP-D-mannose) GDP-D-mannuronic酸。在本文中,我们报告GMD的结构分析有限使用三种不同的蛋白质水解蛋白酶,胰蛋白酶,颔下argc蛋白酶、糜蛋白酶。与这些蛋白酶治疗GMD表示,这种酶的伴部分可以折叠成一个结构域,25日至26日kDa的分子质量。多个蛋白水解乳沟站点存在的羧基末端的领域,表明这段可能代表一个接触区域的蛋白质。最初的蛋白水解作用也生成一个羧基末端片段的分子质量16 - 17 kDa进一步消化成小片段的胰蛋白酶和胰凝乳蛋白酶。蛋白水解乳沟网站被部分伴局部肽片段的测序。参数- 295被认定为初始裂解位点胰蛋白酶和胰凝乳蛋白酶酪氨酸- 278。催化活性的GMD完全废除最初的乳沟。然而,绑定的衬底,GDP-D-mannose,增加稳定性对蛋白水解作用和抑制酶活性的丧失。 GMP and GDP (guanosine 5'-mono- and diphosphates) also blocked the initial cleavage, but NAD and mannose showed no effect. These results suggest that binding of the guanosine moiety at the catalytic site of GMD may induce a conformational change that reduces the accessibility of the cleavage sites to proteases. Binding of [14C]GDP-D-mannose to the amino-terminal domain was not affected by the removal of the carboxyl-terminal 16-kDa fragment. Furthermore, photoaffinity labeling of GMD with [32P]arylazido-beta-alanine-NAD followed by proteolysis demonstrated that the radioactive NAD was covalently linked to the amino-terminal domain. These observations imply that the amino-terminal domain (25-26 kDa) contains both the substrate and cofactor binding sites. However, the carboxyl-terminal fragment (16-17 kDa) may possess amino acid residues essential for catalysis. Thus, proteolysis had little effect on substrate binding, but totally eliminated catalysis. These biochemical data are in complete agreement with amino acid sequence analysis for the existence of substrate and cofactor sites of GMD. A linear peptide map of GMD was constructed for future structure/functional studies.