Ikeda H, Seno E T, Bruton C J, Chater K F
Mol Gen Genet. 1984;196(3):501-7. doi: 10.1007/BF00436199.
Glucose kinase in Streptomyces coelicolor has a molecular weight of about 110,000. In crude extracts, the enzyme exhibited apparent Km values of 0.20 mM for ATP, 0.27 mM for glucose, and 2.2 mM for the glucose analogue 2-deoxyglucose. Mutations (glk) to 2-deoxyglucose-resistance, which greatly reduce glucose kinase activity and result in relief of glucose repression of utilisation of various carbon sources, were mapped between proA and hisA in the S. coelicolor linkage map. Glucose kinase activity, 2-deoxyglucose-sensitivity, glucose utilisation and glucose repression, were all restored to glk mutants by a 3.5 kb DNA fragment cloned from S. coelicolor into a phage vector (phi C31 KC515), and by larger (10-30 kb) fragments cloned into a low copy number plasmid vector (pIJ916). The glk gene was further localised to a 2.9 kb BclI fragment of the cloned DNA by sub-cloning. Part or all of this fragment was present in each of five primary plasmid clones tested.
天蓝色链霉菌中的葡萄糖激酶分子量约为110,000。在粗提物中，该酶对ATP的表观Km值为0.20 mM，对葡萄糖的表观Km值为0.27 mM，对葡萄糖类似物2-脱氧葡萄糖的表观Km值为2.2 mM。对2-脱氧葡萄糖产生抗性的突变（glk）极大地降低了葡萄糖激酶活性，并导致对各种碳源利用的葡萄糖阻遏解除，这些突变在天蓝色链霉菌连锁图谱中的proA和hisA之间定位。通过从天蓝色链霉菌克隆到噬菌体载体（phi C31 KC515）中的3.5 kb DNA片段，以及克隆到低拷贝数质粒载体（pIJ916）中的更大片段（10 - 30 kb），葡萄糖激酶活性、对2-脱氧葡萄糖的敏感性、葡萄糖利用和葡萄糖阻遏都恢复到了glk突变体中。通过亚克隆将glk基因进一步定位到克隆DNA的一个2.9 kb BclI片段上。在测试的五个初级质粒克隆中，每个克隆都含有该片段的部分或全部。
