Department of Biochemistry and Molecular Genetics, University of Illinois, 900 South Ashland Avenue, Chicago, Illinois 60607, United States.
Biochemistry. 2011 Apr 12;50(14):2870-80. doi: 10.1021/bi2001032. Epub 2011 Mar 16.
Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant.
脱氧胞苷激酶 (dCK) 利用 ATP 或 UTP 作为磷酸供体来催化核苷受体的磷酸化。人类 dCK 的动力学特性通过丝氨酸 74 的磷酸化在体内进行调节。该残基是插入区的一部分，远离活性位点。用谷氨酸 (S74E 变体) 取代丝氨酸可以模拟 Ser74 的磷酸化。为了了解磷酸化如何影响 dCK 的催化特性，我们从结构和动力学两方面研究了 dCK 的 S74E 变体。我们观察到，第 74 位的谷氨酸的存在有利于酶采用开放构象。Glu74 通过直接与色氨酸 58 的吲哚侧链相互作用来稳定开放构象，色氨酸 58 是核苷底物碱基附近的残基。开放的 dCK 构象有利于核苷结合，但不利于磷酸转移。相比之下，封闭构象有利于磷酸转移，但不利于产物释放。因此，dCK 必须在催化循环中在开放和封闭状态之间进行转换。我们提出了一个 dCK 的反应方案，其中包括开放和封闭状态之间的转换，这有助于解释野生型 dCK 和 S74E 变体之间观察到的动力学差异。
