Song W J, Jackowski S
Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
J Biol Chem. 1994 Oct 28;269(43):27051-8.
Pantothenate kinase catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis and is regulated by feedback inhibition by CoA. Pantothenate kinase was purified to homogeneity from Escherichia coli and was shown to exist as a homodimer. Kinetic analysis indicated the presence of two ATP binding sites that exhibited positive cooperativity with a Hill coefficient of 1.46. Site-directed mutagenesis of lysine 101 to methionine (K101M) resulted in the inactivation of the enzyme, although dimer formation was not altered. The K101M mutant was unable to bind either adenosine 5'-O-(3-thiotriphosphate) or CoA, supporting the conclusion from kinetic analysis that both the substrate and inhibitor bind to the same site on the enzyme. CoA binding was not cooperative. Coexpression of the K101M mutant gene on a high copy number plasmid in the presence of a chromosomal copy of the wild-type gene resulted in the production of heterodimers between active and inactive subunits. Kinetic analysis of the chimeric heterodimers showed the absence of cooperative ATP interactions and indicated a sequential kinetic mechanism for pantothenate kinase with ATP binding first and pantothenate second. Thus, pantothenate kinase regulation involves the competitive binding of CoA to the ATP site, which blocks ATP binding at one site and prevents positive cooperative ATP binding to the second site on the dimer.
泛酸激酶催化辅酶A（CoA）生物合成中的限速步骤，并受CoA的反馈抑制调节。泛酸激酶从大肠杆菌中纯化至同质，显示以同二聚体形式存在。动力学分析表明存在两个ATP结合位点，它们表现出正协同性，希尔系数为1.46。将赖氨酸101定点突变为甲硫氨酸（K101M）导致酶失活，尽管二聚体形成未改变。K101M突变体无法结合5'-O-（3-硫代三磷酸）腺苷或CoA，支持动力学分析得出的结论，即底物和抑制剂都结合到酶上的同一位点。CoA结合不具有协同性。在野生型基因的染色体拷贝存在的情况下，在高拷贝数质粒上共表达K101M突变体基因导致活性和非活性亚基之间产生异二聚体。对嵌合异二聚体的动力学分析表明不存在协同性ATP相互作用，并表明泛酸激酶的动力学机制是顺序性的，首先是ATP结合，其次是泛酸结合。因此，泛酸激酶调节涉及CoA与ATP位点的竞争性结合，这会阻断一个位点上的ATP结合并阻止ATP与二聚体上的第二个位点进行正协同结合。
