Lillig C H, Schiffmann S, Berndt C, Berken A, Tischka R, Schwenn J D
Biochemistry of Plants, Ruhr-University Bochum, Bochum, 44780, Germany.
Arch Biochem Biophys. 2001 Aug 15;392(2):303-10. doi: 10.1006/abbi.2001.2453.
A cDNA clone (Atakn1) from Arabidopsis thaliana encoding APS-kinase (EC 2.7.1.25) was investigated for structural and catalytic properties of the gene product. Recombinant his10-AtAkn1 formed PAPS at a Vmax of 7.35 U x mg(-1). The Km for APS was 0.14 microM and for ATP 147 microM. APS caused a severe substrate inhibition (K(i) 4.5 microM). The type of inhibition is uncompetitive with respect to MgATP. High ionic strength and reducing thiols stabilized the enzyme activity. Plant APS-kinase is regulated in vitro by the redox charge with thioredoxin as essential activator. Mutagenesis of a serine in S182C and S182F presumed to be involved in the transfer of the phosphoryl group had no effect upon catalytic activity. Using a yeast two-hybrid system with AtAkn1 as bait, an interacting clone was detected from a cDNA library of A. thaliana cv. Columbia that codes for an APS-kinase iso-form (Atakn2). Complementation of APS-kinase-deficient Saccharomyces cerevisiae met14 showed that AtAkn2 is functionally active as APS-kinase. It was immunologically related to AtAkn1 and presumably represents a plastidal iso-form of the plant APS-kinase gene family.
对来自拟南芥的一个编码APS激酶（EC 2.7.1.25）的cDNA克隆（Atakn1）进行了基因产物的结构和催化特性研究。重组his10-AtAkn1以7.35 U x mg(-1)的Vmax形成PAPS。APS的Km为0.14 microM，ATP的Km为147 microM。APS引起严重的底物抑制（K(i) 4.5 microM）。这种抑制类型对MgATP而言是非竞争性的。高离子强度和还原型硫醇可稳定酶活性。植物APS激酶在体外受氧化还原电荷调节，硫氧还蛋白是必需的激活剂。推测参与磷酰基转移的S182C和S182F中丝氨酸的诱变对催化活性没有影响。以AtAkn1为诱饵，利用酵母双杂交系统从拟南芥cv. Columbia的cDNA文库中检测到一个相互作用的克隆，其编码一种APS激酶同工型（Atakn2）。对APS激酶缺陷型酿酒酵母met14的互补作用表明，AtAkn2作为APS激酶具有功能活性。它与AtAkn1有免疫相关性，可能代表植物APS激酶基因家族的一种质体同工型。
