Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase: rapid phosphoribosyl transfer chemistry.
Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase: rapid phosphoribosyl transfer chemistry.
复制标题
人次黄嘌呤鸟嘌呤磷酸核糖基转移酶的动力学机制：快速磷酸核糖基转移化学。
DOI：
10.1021/bi9616007
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Grubmeyer,C
中科院分区：
文献类型：
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作者：
Xu,Y;Eads,J;Sacchettini,JC;Grubmeyer,C
次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HGPRTase）是Lesch-Nyhan综合征的发病部位，是前药6-巯基嘌呤和别嘌呤醇的激活剂，也是抗寄生虫化疗的靶点。与GMP复合的重组人酶的三维结构最近已被解决[埃兹，J.，Scapin，G.，徐，Y.，Grubmeyer，C.，& Sacchettini，J. C.（1994）Cell 78，325 - 334]。在这里，配体结合，预稳态动力学，同位素捕获，和同位素交换实验详细的顺序动力学机制的酶。在正向反应中，碱基（次黄嘌呤或鸟嘌呤）与PRPP反应形成单磷酸核苷和PPi，PRPP的结合先于碱基的结合，而在反向反应中，IMP首先结合。与tokcat相比，磷酸核糖基在正向（131 vs 6.0 s-1）和反向（9 vs 0.17 s-1）方向上的转移都很快。在正向方向上，产物焦磷酸盐快速解离（>12 s-1），随后释放IMP（6.0 s-1）。在相反的方向上，Hx解离迅速（9.5 s-1）和PRPP解离缓慢（0.24 s-1）。在正向反应中，鸟嘌呤的利用率比次黄嘌呤快，这是GMP产物释放更快的结果，而不是化学步骤速率不同的结果。快速化学反应和缓慢产物解离的动力学机制解释了先前观察到的替代产物鸟嘌呤刺激而非抑制IMP焦磷酸解的能力。次黄嘌呤磷酸核糖转移反应的总体平衡远离核苷酸产物（Keq = 1.6 × 105），处于PRPP连接核苷酸形成的高端。HGPRTase·IMP复合物的三维结构已解析至2.4 μ m分辨率，与GMP复合物同晶。的配体结合和动力学研究的结果进行了讨论，在光的结构数据。
Hypoxanthine−guanine phosphoribosyltransferase (HGPRTase) is the locus of Lesch-Nyhan syndrome, the activator of the prodrugs 6-mercaptopurine and allopurinol, and a target for antiparasitic chemotherapy. The three-dimensional structure of the recombinant human enzyme in complex with GMP has recently been solved [Eads, J., Scapin, G., Xu, Y., Grubmeyer, C., & Sacchettini, J. C. (1994)Cell 78, 325−334]. Here, ligand binding, pre-steady state kinetics, isotope trapping, and isotope exchange experiments are presented which detail the sequential kinetic mechanism of the enzyme. In the forward reaction, in which a base (hypoxanthine or guanine) reacts with PRPP to form nucleoside monophosphate and PPi, binding of PRPP precedes that of the base, and in the reverse direction, IMP binds first. Compared tokcat, phosphoribosyl group transfer is rapid in both the forward (131 vs 6.0 s-1) and reverse (9 vs 0.17 s-1) directions. In the forward direction, product pyrophosphate dissociates rapidly (>12 s-1) followed by release of IMP (6.0 s-1). In the reverse direction, Hx dissociates rapidly (9.5 s-1) and PRPP dissociates slowly (0.24 s-1). The more rapid rate of utilization of guanine than hypoxanthine in the forward reaction is the result of the faster release of product GMP rather than the result of differences in the rate of the chemical step. The kinetic mechanism, with rapid chemistry and slow product dissociation, accounts for the previously observed ability of the alternative product guanine to stimulate, rather than inhibit, the pyrophosphorolysis of IMP. The overall equilibrium for the hypoxanthine phosphoribosyl transfer reaction lies far toward nucleotide product (Keq≈ 1.6 × 105), at the high end for PRPP-linked nucleotide formation. The three-dimensional structure of the HGPRTase·IMP complex has been solved to 2.4 Å resolution and is isomorphous with the GMP complex. The results of the ligand binding and kinetic studies are discussed in light of the structural data.