Hughes T L, Hahn T M, Reynolds K K, Shewach D S
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109, USA.
Biochemistry. 1997 Jun 17;36(24):7540-7. doi: 10.1021/bi970059r.
Deoxycytidine kinase is the rate-limiting process in the activation for several clinically important antitumor agents. Previous studies have focused on deoxycytidine (dCyd) and adenosine triphosphate (ATP) as substrates for this enzyme. In view of recent data indicating that uridine triphosphate (UTP) is the physiologic phosphate donor for this enzyme, a study of the kinetic properties of dCyd kinase with dCyd and UTP was undertaken. The results presented here demonstrate that UTP and ATP produce kinetically distinguishable differences in nucleoside phosphorylation by dCyd kinase. At high dCyd concentrations, dCyd kinase exhibited substrate activation with ATP. In contrast, in the presence of UTP, substrate inhibition was observed at concentrations of dCyd greater than 3 microM. Inhibition by dCyd was noncompetitive with respect to UTP and could not be reversed by a 200-fold increase in UTP concentration, indicating that the inhibition was not due to dCyd binding at the nucleotide binding site. The kinetic mechanism for dCyd kinase was determined with dCyd and UTP as substrates. UTP was the preferred phosphate donor with a true Km value of 1 microM compared to 54 microM with ATP, resulting in a 50-fold greater substrate efficiency for UTP. Although the double-reciprocal plots with UTP produced parallel lines, initial velocity plots with other phosphate donors and product inhibition studies indicated that dCyd kinase formed a ternary complex with its substrates. The parallel lines with UTP were apparently due to a low dissociation constant for UTP, which was calculated as more than 13-fold lower than its Km value. Analysis of product inhibition studies indicated that dCyd kinase followed an ordered A-B random P-Q reaction sequence, with UTP as the first substrate to bind. In contrast, previous results demonstrated a random bi-bi sequence for dCyd kinase in the presence of ATP. The combined results indicate that the enzyme can follow a random bi-bi reaction sequence, but with UTP as the phosphate donor, the addition of nucleotide prior to dCyd is strongly preferred. The noncompetitive substrate inhibition, which was independent of UTP concentration, indicates that high concentrations of dCyd promote addition of the nucleoside prior to UTP, resulting in a lower velocity.
脱氧胞苷激酶是几种临床上重要的抗肿瘤药物激活过程中的限速环节。以往的研究主要聚焦于脱氧胞苷（dCyd）和三磷酸腺苷（ATP）作为该酶的底物。鉴于最近的数据表明三磷酸尿苷（UTP）是该酶的生理性磷酸供体，因此开展了一项关于dCyd激酶与dCyd和UTP的动力学特性的研究。此处呈现的结果表明，UTP和ATP在dCyd激酶介导的核苷磷酸化过程中产生了动力学上可区分的差异。在高dCyd浓度下，dCyd激酶对ATP表现出底物激活作用。相反，在UTP存在的情况下，当dCyd浓度大于3 microM时观察到底物抑制现象。dCyd的抑制作用对UTP而言是非竞争性的，并且UTP浓度增加200倍也无法逆转这种抑制作用，这表明该抑制作用并非由于dCyd在核苷酸结合位点的结合所致。以dCyd和UTP作为底物确定了dCyd激酶的动力学机制。UTP是首选磷酸供体，其真实Km值为1 microM，而ATP的Km值为54 microM，这使得UTP的底物效率高出50倍。尽管与UTP的双倒数图产生了平行线，但与其他磷酸供体的初速度图以及产物抑制研究表明，dCyd激酶与其底物形成了三元复合物。与UTP的平行线显然是由于UTP的解离常数较低，经计算其比Km值低13倍以上。产物抑制研究分析表明，dCyd激酶遵循有序的A - B随机P - Q反应序列，其中UTP是第一个结合的底物。相反，先前的结果表明在ATP存在下dCyd激酶遵循随机双底物双产物序列。综合结果表明，该酶可以遵循随机双底物双产物反应序列，但以UTP作为磷酸供体时，强烈优先于dCyd先添加核苷酸。与UTP浓度无关的非竞争性底物抑制表明，高浓度的dCyd会促进在UTP之前添加核苷，从而导致速度降低。
