Larsson G, Nyman P O, Kvassman J O
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden.
J Biol Chem. 1996 Sep 27;271(39):24010-6. doi: 10.1074/jbc.271.39.24010.
The enzyme dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, thereby preventing a deleterious incorporation of uracil into DNA. The best known dUTPase is that from Escherichia coli, which, like the human enzyme, consists of three identical subunits. In the present work, the catalytic properties of the E. coli dUTPase were investigated in the pH range 5-11. The enzyme was found to be highly specific for dUTP and discriminated both base and sugar as well as the phosphate moiety (bound dUDP was not hydrolyzed). The second best substrate among the nucleotides serving as building blocks for DNA was dCTP, which was hydrolyzed an astonishing 10(5) times less efficiently than dUTP, a decline largely accounted for by a higher Km for dCTP. With dUTP.Mg as substrate, kcat was found to vary little with pH and to range from 6 to 9 s-1. Km passed through a broad minimum in the neutral pH range with values approaching 10(-7) M. It increased with deprotonation of the uracil moiety of dUTP and showed dependence on two ionizations in the enzyme, exhibiting pKa values of 5.8 and 10.3. When excess dUTPase was reacted with dUTP middle dotMg at pH 8, the two protons transferred to the reaction medium were released in a concerted mode after the rate-limiting step. The Mg2+ ion enhances binding to dUTPase of dUTP by a factor of 100 and dUDP by a factor of 10. Only one enantiomer of the substrate analog 2'-deoxyuridine-5'-(alpha-thio)-triphosphate was hydrolyzed by the enzyme. These results are interpreted to favor a catalytic mechanism involving magnesium binding to the alpha-phosphate, rate-limiting hydrolysis by a shielded and activated water molecule and a fast ordered desorption of the products. The results are discussed with reference to recent data on the structure of the E. coli dUTPase.UDP complex.
dUTP酶催化dUTP水解生成dUMP和焦磷酸，从而防止尿嘧啶有害地掺入DNA中。最著名的dUTP酶来自大肠杆菌，它与人类酶一样，由三个相同的亚基组成。在本研究中，研究了大肠杆菌dUTP酶在pH值5 - 11范围内的催化特性。发现该酶对dUTP具有高度特异性，能区分碱基、糖以及磷酸基团（结合的dUDP不被水解）。作为DNA组成单元的核苷酸中，第二好的底物是dCTP，其水解效率比dUTP低惊人的10⁵倍，这种下降主要是由于dCTP的Km值较高。以dUTP·Mg作为底物时，发现kcat随pH值变化不大，范围为6至9 s⁻¹。Km在中性pH范围内有一个较宽的最小值，数值接近10⁻⁷ M。它随着dUTP尿嘧啶部分的去质子化而增加，并显示出依赖于酶中的两个电离，pKa值分别为5.8和10.3。当过量的dUTP酶在pH 8下与dUTP·Mg反应时，转移到反应介质中的两个质子在限速步骤之后以协同模式释放。Mg²⁺离子使dUTP与dUTP酶的结合增强100倍，使dUDP与dUTP酶的结合增强10倍。该酶仅水解底物类似物2'-脱氧尿苷-5'-(α-硫代)-三磷酸的一种对映体。这些结果被解释为支持一种催化机制，该机制涉及镁与α-磷酸的结合、由屏蔽和活化的水分子进行限速水解以及产物的快速有序解吸。结合最近关于大肠杆菌dUTP酶 - UDP复合物结构的数据对这些结果进行了讨论。
