Blanche F, Debussche L, Famechon A, Thibaut D, Cameron B, Crouzet J
Département Analyse, Institut des Biotechnologies, Vitry-sur-Seine, France.
J Bacteriol. 1991 Oct;173(19):6052-7. doi: 10.1128/jb.173.19.6052-6057.1991.
The two consecutive activities of the cobalamin biosynthetic pathway that catalyze the conversion of cobinamide to cobinamide phosphate (cobinamide kinase) and of cobinamide phosphate to GDP-cobinamide (cobinamide phosphate guanylytransferase) were shown to be carried by the same protein in Pseudomonas denitrificans. This bifunctional protein was purified to homogeneity by high-performance liquid chromatography of extracts of a recombinant strain of this microorganism, and the sequence of the first 10 amino acid residues at the N terminus was determined. Both activities were specific to the coenzyme forms of the corrinoid substrates and exhibited an optimum pH at 8.8. Both ATP and GTP were shown to be in vitro gamma-phosphate donors for cobinamide kinase. However, competition experiments demonstrated that ATP was the preferred substrate, a result that can be explained in terms of the kinetic properties of the enzyme. Labeling experiments established that the phosphate group of cobinamide phosphate is quantitatively retained as the inner phosphate of GDP-cobinamide during the guanylyltransferase reaction. The native protein had an apparent molecular weight of 40,000, as estimated by gel filtration, and consisted of two identical subunits of Mr 20,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein had an isoelectric point of 5.35 and contained a high-affinity GTP-binding site (Kaff.(GTP) = 0.22 microM). Binding of GTP onto this site resulted in a marked increase of the affinity of cobinamide kinase for cobinamide. This property and other kinetic properties may regulate the enzyme and prevent the accumulation of cobinamide phosphate.
在反硝化假单胞菌中，钴胺素生物合成途径中催化钴胺酰胺转化为磷酸钴胺酰胺（钴胺酰胺激酶）以及磷酸钴胺酰胺转化为GDP-钴胺酰胺（磷酸钴胺酰胺鸟苷酰转移酶）的两个连续反应，被证明是由同一种蛋白质催化的。通过对该微生物重组菌株提取物进行高效液相色谱法，将这种双功能蛋白纯化至同质，并测定了其N端前10个氨基酸残基的序列。这两种活性对类咕啉底物的辅酶形式具有特异性，并且在pH 8.8时表现出最佳活性。ATP和GTP在体外均被证明是钴胺酰胺激酶的γ-磷酸供体。然而，竞争实验表明ATP是首选底物，这一结果可以根据该酶的动力学特性来解释。标记实验证实，在鸟苷酰转移酶反应过程中，磷酸钴胺酰胺的磷酸基团作为GDP-钴胺酰胺的内部磷酸基团被定量保留。通过凝胶过滤估计，天然蛋白的表观分子量为40,000，而通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，其由两个分子量为20,000的相同亚基组成。该蛋白的等电点为5.35，并且含有一个高亲和力的GTP结合位点（Kaff.(GTP) = 0.22 microM）。GTP与该位点的结合导致钴胺酰胺激酶对钴胺酰胺的亲和力显著增加。这一特性和其他动力学特性可能对该酶起到调节作用，并防止磷酸钴胺酰胺的积累。
