Luo Ruibai, Ahvazi Bijan, Amariei Diana, Shroder Deborah, Burrola Beatriz, Losert Wolfgang, Randazzo Paul A
Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
Biochem J. 2007 Mar 15;402(3):439-47. doi: 10.1042/BJ20061217.
Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a k(cat) of 57+/-5 s(-1) and a K(m) of 2.2+/-0.5 microM determined by steady-state kinetics and a kcat of 56+/-7 s(-1) determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4-), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than K(m). Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the k(cat). Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP-ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.
Arf（ADP核糖基化因子）GAPs（GTP酶激活蛋白）是催化与小GTP结合蛋白Arf结合的GTP水解的酶。它们也被认为可作为Arf效应器和癌基因发挥作用。我们已着手使用截短形式的ASAP1 [具有SH3（Src同源结构域3）结构域、锚蛋白重复序列和PH（普列克底物蛋白同源）结构域1的Arf GAP]作为模型来表征GAP诱导的GTP水解动力学。我们发现，ASAP1将Arf1-GTP用作底物，通过稳态动力学测定的k(cat)为57±5 s(-1)，K(m)为2.2±0.5 microM，通过单周转动力学测定的kcat为56±7 s(-1)。四氟铝酸盐（AlF4-）可稳定其他Ras家族成员与其同源GAP的复合物，它也稳定了Arf1-GDP与ASAP1的复合物。正如预期的那样，将Arg-497突变为赖氨酸残基对kcat的影响远大于对K(m)的影响。根据先前的研究预测，改变Trp-479、Iso-490、Arg-505、Leu-511或Asp-512会影响对Arf1-GTP的亲和力。相反，这些突变主要影响k(cat)。在体外缺乏活性的突变体在ASAP1功能的体内测定（背侧褶皱形成的抑制）中同样缺乏活性。我们的结果支持以下结论：Arf GAP ASAP1与Arf1-GTP以二元复合物形式发挥作用，诱导向GTP水解的过渡态。这些结果使我们推测，Arf1-GTP-ASAP1从基态转变为催化活性状态时会发生显著的构象变化。文中讨论了ASAP1假定效应器功能的影响。
