Blaszczyk Jaroslaw, Li Yue, Wu Yan, Shi Genbin, Ji Xinhua, Yan Honggao
Macromolecular Crystallography Laboratory, National Cancer Institute, P.O. Box B, Frederick, Maryland 21702, USA.
Biochemistry. 2004 Feb 17;43(6):1469-77. doi: 10.1021/bi036053l.
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphoryl group from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP) following an ordered bi-bi mechanism with ATP as the first substrate. The rate-limiting step of the reaction is product release, and the complete active center is assembled and sealed only upon the binding of both ATP and HP. The assembly of the active center involves large conformational changes in three catalytic loops, among which loop 3 undergoes the most dramatic and unusual changes. To investigate the roles of loop 3 in catalysis, we have made a deletion mutant, which has been investigated by biochemical and X-ray crystallographic analysis. The biochemical data showed that the deletion mutation does not have significant effects on the dissociation constants or the rate constants for the binding of the first substrate MgATP or its analogues. The dissociation constant of HP for the mutant increases by a factor of approximately 100, which is due to a large increase in the dissociation rate constant. The deletion mutation causes a shift of the rate-limiting step in the reaction and a decrease in the rate constant for the chemical step by a factor of approximately 1.1 x 10(5). The crystal structures revealed that the deletion mutation does not affect protein folding, but the catalytic center of the mutant is not fully assembled even upon the formation of the ternary complex and is not properly sealed. The results together suggest that loop 3 is dispensable for the folding of the protein and the binding of the first substrate MgATP, but is required for the assembling and sealing of the active center. The loop plays an important role in the stabilization of the ternary complex and is critical for catalysis.
6-羟甲基-7,8-二氢蝶呤焦磷酸激酶（HPPK）按照有序双底物机制，以ATP作为第一个底物，催化焦磷酸基团从ATP转移至6-羟甲基-7,8-二氢蝶呤（HP）。该反应的限速步骤是产物释放，并且只有在ATP和HP都结合后，完整的活性中心才会组装并封闭。活性中心的组装涉及三个催化环的大构象变化，其中环3经历了最显著和异常的变化。为了研究环3在催化中的作用，我们构建了一个缺失突变体，并通过生化和X射线晶体学分析对其进行了研究。生化数据表明，缺失突变对第一个底物MgATP或其类似物结合的解离常数或速率常数没有显著影响。突变体对HP的解离常数增加了约100倍，这是由于解离速率常数大幅增加所致。缺失突变导致反应中的限速步骤发生偏移，化学步骤的速率常数降低了约1.1×10⁵倍。晶体结构显示，缺失突变不影响蛋白质折叠，但即使在三元复合物形成后，突变体的催化中心也未完全组装，且未正确封闭。这些结果共同表明，环3对于蛋白质折叠和第一个底物MgATP的结合是可有可无的，但对于活性中心的组装和封闭是必需的。该环在三元复合物的稳定中起重要作用，对催化至关重要。
