Ménard L, Tomhave E, Casey P J, Uhing R J, Snyderman R, Didsbury J R
Department of Medicine, Duke University Medical Center, Durham, NC 27710.
Eur J Biochem. 1992 Jun 1;206(2):537-46. doi: 10.1111/j.1432-1033.1992.tb16957.x.
Rac1, a member of the family of low-molecular-mass GTP-binding proteins, functions in phagocytic leukocytes as a component necessary for activation of the respiratory burst. To characterize the biochemical properties of rac1, the protein was expressed as a fusion protein in Escherichia coli and purified to greater than 99% homogeneity by affinity chromatography. Rac1 protein bound maximally bound and hydrolyzed GTP under low free-Mg2+ concentrations. Under those conditions, (45 nm free Mg2+), purified rac1 exhibited a steady-state GTPase activity of 18 nmol.min-1.mg protein-1 (turnover number approximately 0.39 min-1 at 37 degrees C), which is 40-fold higher than H-ras. The high intrinsic GTPase activity of rac1 under low free Mg2+ was mainly due to an increased kcat, the rate constant for hydrolysis of bound GTP, which was 0.29 min-1 for rac1 vs 0.007 min-1 for H-ras (at 20 degrees C). Rac1 also released bound GDP faster than H-ras (koff.GDP = 1.02 min-1 for rac1 vs 0.33 min-1 for H-ras at 20 degrees C). In contrast, rac1 released bound guanosine 5'-[gamma-thio]triphosphate (GTP[S]) at a slower rate than H-ras (koff.GTP[S] approximately 0.04 min-1 for rac1 vs 0.31 min-1 for H-ras at 20 degrees C). Rac1 was a very good substrate for in vitro geranylgeranylation (C20) but not for farnesylation (C15), whereas the converse is true for H-ras. Surprisingly, rac1 was a very poor substrate for in vitro ADP-ribosylation by the C3 component of Clostridium botulinum toxin compared to rhoA. As a further characterization of rac1, a mutant was made in which the Thr115 was replaced by asparagine. This protein (referred to as [Thr115----Asn]rac1) contains the consensus amino acids of all four GTP-binding domains of H-ras. The koff.GDP of [Thr115----Asn]rac1 was reduced to that of H-ras, but [Thr115----Asn]rac1 exhibited essentially identical kcat (0.13 min-1 at 20 degrees C) and koff-GTP[S] (0.03 min-1 at 20 degrees C) values as the wild-type protein. Thus, the region(s) in rac1 which control the dissociation of GTP[S] (and presumably GTP) do not entirely coincide with those controlling GDP dissociation. Biochemical analysis of [Thr115----Asn]rac1 also suggests that the region responsible for the increased kcat of rac1 is not within the consensus amino acids of the four guanine-nucleotide-binding domains.
Rac1是低分子量GTP结合蛋白家族的成员之一，在吞噬性白细胞中作为呼吸爆发激活所必需的成分发挥作用。为了表征Rac1的生化特性，该蛋白在大肠杆菌中作为融合蛋白表达，并通过亲和色谱法纯化至大于99%的纯度。Rac1蛋白在低游离Mg2+浓度下最大程度地结合并水解GTP。在这些条件下（45 nM游离Mg2+），纯化的Rac1表现出18 nmol·min-1·mg蛋白-1的稳态GTP酶活性（37℃时周转数约为0.39 min-1），这比H-ras高40倍。Rac1在低游离Mg2+下的高内在GTP酶活性主要归因于kcat的增加，即结合GTP水解的速率常数，Rac1为0.29 min-1，而H-ras为0.007 min-1（20℃时）。Rac1释放结合的GDP也比H-ras快（20℃时，Rac1的koff.GDP = 1.02 min-1，H-ras为0.33 min-1）。相比之下，Rac1释放结合的鸟苷5'-[γ-硫代]三磷酸（GTP[S]）的速率比H-ras慢（20℃时，Rac1的koff.GTP[S]约为0.04 min-1，H-ras为0.31 min-1）。Rac1是体外香叶基香叶基化（C20）的良好底物，但不是法尼基化（C15）的底物，而H-ras则相反。令人惊讶的是，与rhoA相比，Rac1是肉毒杆菌毒素C3成分体外ADP核糖基化的非常差的底物。作为对Rac1的进一步表征，构建了一个将Thr115替换为天冬酰胺的突变体。该蛋白（称为[Thr115→Asn]Rac1）包含H-ras所有四个GTP结合结构域的共有氨基酸。[Thr115→Asn]Rac1的koff.GDP降低至H-ras的水平，但[Thr115→Asn]Rac1表现出与野生型蛋白基本相同的kcat（20℃时为0.13 min-1）和koff-GTP[S]（20℃时为0.03 min-1）值。因此，Rac1中控制GTP[S]（可能还有GTP）解离的区域与控制GDP解离的区域并不完全一致。对[Thr115→Asn]Rac1的生化分析还表明，负责Rac1的kcat增加的区域不在四个鸟嘌呤核苷酸结合结构域的共有氨基酸内。
