Richter M F, Schwemmle M, Herrmann C, Wittinghofer A, Staeheli P
Abteilung Virologie, Universität Freiburg, Germany.
J Biol Chem. 1995 Jun 2;270(22):13512-7.
MxA is a GTPase encoded by an interferon-activated human gene which inhibits the multiplication of several RNA viruses. Recombinant histidine-tagged MxA protein (His-MxA) was expressed in Escherichia coli and purified to near homogeneity. Gel filtration showed that it formed high molecular weight oligomers. Purified His-MxA exhibited specific GTP hydrolysis rates of up to 350 nmol of GTP/min/mg of protein, corresponding to a turnover number of 27 min-1. The Km for this reaction was 260 microM. Guanine nucleotides did not copurify with His-MxA. Binding experiments in solution with fluorescent-labeled nucleotides confirmed that His-MxA binds guanine nucleotides rather weakly and further showed that the fluorescent GDP analog N-methylanthraniloyl (mant)-GDP had a much lower affinity for His-MxA (Kd 20 microM, koff 8.5 s-1) than the nonhydrolyzable GTP analog mant-5'-guanylyl-beta,gamma-imidotriphosphate (mant-GMP-PNP) (Kd 0.75 microM, koff 0.012 s-1). Competitive binding studies with nonlabeled nucleotides revealed a similar binding preference of His-MxA for GTP over GDP: the Kd for GTP was 20 microM, whereas the Kd for GDP was 100 microM. Thus, a high percentage of MxA molecules may be complexed with GTP in vivo.
MxA是一种由干扰素激活的人类基因编码的GTP酶，可抑制多种RNA病毒的增殖。重组组氨酸标签的MxA蛋白（His-MxA）在大肠杆菌中表达并纯化至接近均一。凝胶过滤显示它形成了高分子量的寡聚体。纯化的His-MxA表现出高达350 nmol GTP/分钟/毫克蛋白的特异性GTP水解速率，对应于27分钟-1的周转数。该反应的Km为260 μM。鸟嘌呤核苷酸不与His-MxA共纯化。用荧光标记核苷酸进行的溶液结合实验证实His-MxA与鸟嘌呤核苷酸的结合相当弱，并进一步表明荧光GDP类似物N-甲基邻氨基苯甲酰基（mant）-GDP对His-MxA的亲和力（Kd 20 μM，koff 8.5 s-1）远低于不可水解的GTP类似物mant-5'-鸟苷酰基-β，γ-亚氨基三磷酸（mant-GMP-PNP）（Kd 0.75 μM，koff 0.012 s-1）。用未标记核苷酸进行的竞争性结合研究揭示了His-MxA对GTP的结合偏好高于GDP：GTP的Kd为20 μM，而GDP的Kd为100 μM。因此，体内可能有高比例的MxA分子与GTP复合。
