Nakai H, Richardson C C
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1990 Mar 15;265(8):4411-9.
Escherichia coli encodes a dGTP triphosphohydrolase (dGTPase) that cleaves dGTP to deoxyguanosine and tripolyphosphate. dGTP is hydrolyzed with a Michaelis constant (Km) of 5 microM and a maximal velocity (Vmax) of 1.8 mumols/min/mg. The ribonucleotide GTP is a poor substrate with a much lower affinity. It is hydrolyzed with a Km of 150 microM and Vmax of 0.07 mumols/min/mg. Bacteriophage T7 encodes a specific inhibitor of dGTPase, the gene 1.2 protein, that forms a tight complex with the enzyme. The enzyme-inhibitor complex binds dGTP with a dissociation constant (KD) of 1.5 microM, but the bound dGTP is not hydrolyzed. It remains stably bound to the complex with a half-life of approximately 5 min. In contrast, dGTP is unable to bind to gene 1.2 protein alone, and dGTP bound to dGTPase alone is quickly hydrolyzed and released. Surprisingly, the dGTPase-gene 1.2 protein complex has a higher affinity for GTP than for dGTP. GTP is stably bound to the dGTPase-gene 1.2 protein complex with a half-life greater than 30 min and KD of 0.8 microM; GTP is not stably bound to either dGTPase or gene 1.2 protein alone. Both GTP and dGTP bind to and stabilize the dGTPase-gene 1.2 protein complex, inhibiting its dissociation. Although the presence of dGTP induces conformation changes in dGTPase so that it is unable to associate with the gene 1.2 protein, saturating concentrations of GTP have no such effect. The enzyme efficiently associates with its inhibitor in the presence of GTP. These results indicate that E. coli dGTPase and gene 1.2 protein interact to form a high affinity GTP-binding site. dGTP is most effective in preventing the association of the enzyme with the inhibitor whereas GTP is most effective in preventing the dissociation of the enzyme-inhibitor complex.
大肠杆菌编码一种dGTP三磷酸水解酶（dGTP酶），该酶可将dGTP裂解为脱氧鸟苷和三聚磷酸。dGTP水解的米氏常数（Km）为5微摩尔，最大反应速度（Vmax）为1.8微摩尔/分钟/毫克。核糖核苷酸GTP是一种较差的底物，亲和力低得多。它水解的Km为150微摩尔，Vmax为0.07微摩尔/分钟/毫克。噬菌体T7编码一种dGTP酶的特异性抑制剂，即基因1.2蛋白，它与该酶形成紧密复合物。酶-抑制剂复合物以1.5微摩尔的解离常数（KD）结合dGTP，但结合的dGTP不被水解。它以约5分钟的半衰期稳定地结合在复合物上。相比之下，dGTP不能单独与基因1.2蛋白结合，单独与dGTP酶结合的dGTP会迅速水解并释放。令人惊讶的是，dGTP酶-基因1.2蛋白复合物对GTP的亲和力高于对dGTP的亲和力。GTP以大于30分钟的半衰期和0.8微摩尔的KD稳定地结合在dGTP酶-基因1.2蛋白复合物上；GTP不能单独稳定地结合在dGTP酶或基因1.2蛋白上。GTP和dGTP都能结合并稳定dGTP酶-基因1.2蛋白复合物，抑制其解离。尽管dGTP的存在会诱导dGTP酶的构象变化，使其无法与基因1.2蛋白结合，但饱和浓度的GTP没有这种作用。在GTP存在的情况下，该酶能有效地与抑制剂结合。这些结果表明，大肠杆菌dGTP酶和基因1.2蛋白相互作用形成一个高亲和力的GTP结合位点。dGTP在阻止酶与抑制剂结合方面最有效，而GTP在阻止酶-抑制剂复合物解离方面最有效。
