Zhang B, Zheng Y
Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163, USA.
Biochemistry. 1998 Apr 14;37(15):5249-57. doi: 10.1021/bi9718447.
The small GTP-binding protein RhoA becomes inactivated by hydrolyzing bound GTP to GDP through its intrinsic GTPase activity which is further stimulated by a family of Rho GTPase-activating proteins (GAPs). Here we have compared the kinetics of interaction between recombinant RhoA and the RhoGAP domains of p190, p50RhoGAP, Bcr, and 3BP-1. The intrinsic rate of GTP hydrolysis by RhoA is relatively slow when compared to other Rho-family GTPases such as Cdc42 or Rac1 with a rate constant of 0.022 min-1, which can be further stimulated at least 4000-fold by p190 or p50RhoGAP. The RhoGAP domains of Bcr and 3BP-1, which were thought to be inactive toward RhoA, are also found capable of stimulating the GTPase activity of RhoA in a dose-dependent manner. The supreme catalytic activities of p190 and p50RhoGAP toward RhoA reside mostly in their lower Km values (1.79 and 2.83 microM, respectively) which correlate well with their binding affinity for GMP-PNP-bound RhoA (2.18 and 2. 47 microM, respectively), in contrast with Bcr and 3BP-1 which interact with the activated RhoA with much higher Km (89 microM). However, the mechanisms of catalysis by p190 and p50RhoGAP are distinct in at least three aspects: (1) p50RhoGAP displays an effect of product inhibition by binding to the GDP-bound form of RhoA with a Kd of 6 microM in comparison with the Kd for p190 of 33 microM; (2) the Km of p190 increases drastically upon the increase of salt and Mg2+ concentrations, conditions under which only modest changes of Km for p50RhoGAP are observed; and (3) p50RhoGAP remains partially active toward the effector domain mutants of RhoA, Y34K, and T37A, whereas p190 is completely inactive toward Y34K and T37A. These results suggest that there exists a unique mechanism of functional interaction between RhoA and individual RhoGAP which involves distinct structural determinants of the small G-protein to cause the apparent differences in kinetic properties.
小GTP结合蛋白RhoA通过其内在的GTP酶活性将结合的GTP水解为GDP而失活，而一类Rho GTP酶激活蛋白（GAPs）可进一步刺激该活性。在此，我们比较了重组RhoA与p190、p50RhoGAP、Bcr和3BP-1的RhoGAP结构域之间的相互作用动力学。与其他Rho家族GTP酶如Cdc42或Rac1相比，RhoA的内在GTP水解速率相对较慢，速率常数为0.022 min-1，p190或p50RhoGAP可将其进一步刺激至少4000倍。Bcr和3BP-1的RhoGAP结构域，原本被认为对RhoA无活性，现在也发现能够以剂量依赖的方式刺激RhoA的GTP酶活性。p190和p50RhoGAP对RhoA的最高催化活性主要在于它们较低的Km值（分别为1.79和2.83 microM），这与它们对GMP-PNP结合的RhoA的结合亲和力（分别为2.18和2.47 microM）密切相关，而Bcr和3BP-1与活化的RhoA相互作用时的Km值要高得多（89 microM）。然而，p190和p50RhoGAP的催化机制至少在三个方面有所不同：（1）p50RhoGAP通过与GDP结合形式的RhoA结合表现出产物抑制作用，Kd为6 microM，而p190的Kd为33 microM；（2）随着盐和Mg2+浓度的增加，p190的Km急剧增加，而在这些条件下，仅观察到p50RhoGAP的Km有适度变化；（3）p50RhoGAP对RhoA的效应结构域突变体Y34K和T37A仍有部分活性，而p190对Y34K和T37A完全无活性。这些结果表明，RhoA与单个RhoGAP之间存在独特的功能相互作用机制，其中涉及小G蛋白的不同结构决定因素，从而导致动力学性质上的明显差异。
