Burch T J, Haas A L
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Biochemistry. 1994 Jun 14;33(23):7300-8. doi: 10.1021/bi00189a035.
The strict evolutionary conservation of ubiquitin suggests an essential role for each residue in the folding, stability, or function of the protein but precludes identification of such contributions through interspecies comparison of ubiquitin sequences. However, site-directed mutagenesis potentially allows assignment of specific function(s) for each residue. The four arginines present on ubiquitin at positions 42, 54, 72, and 74 were independently mutated to leucine and their effects on the interaction of the resulting polypeptides with ubiquitin-activating enzyme (E1) were characterized. All of the mutants except UbR54L exhibited altered kinetics for E1-catalyzed ATP:PPi exchange compared to wild-type ubiquitin. In addition, the UbR72L mutant altered the mechanism of E1 from strictly order addition of substrates to random addition with respect to ATP and ubiquitin. Values for the intrinsic Kd of ubiquitin binding were determined by coupling the net forward reaction of E1 to the E232K-catalyzed conjugation of histone H2B. Only R54 and R72 residues participate in the initial binding of free ubiquitin, resulting in a 6- or 58-fold increase in Kd for UbR54L or UbR72L, respectively, compared to wild type. More significant effects of the UbR42L and UbR72L mutants were observed for binding of their respective ubiquitin adenylate intermediates within the E1 active site. Wild-type ubiquitin adenylate binds to E1 with an estimated Kd < or = 8 x 10(-12) M while intermediates formed with UbR42L or UbR72L each bind with ca. 10(3)-fold lower affinity, representing a destabilization of > or = 7 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)
泛素在进化上的严格保守性表明，每个残基在该蛋白质的折叠、稳定性或功能中都起着至关重要的作用，但通过泛素序列的种间比较无法确定这些残基的具体贡献。然而，定点诱变有可能确定每个残基的特定功能。泛素第42、54、72和74位上的四个精氨酸被分别突变为亮氨酸，并对所得多肽与泛素激活酶（E1）相互作用的影响进行了表征。与野生型泛素相比，除UbR54L外的所有突变体在E1催化的ATP:PPi交换反应中均表现出动力学改变。此外，UbR72L突变体改变了E1的反应机制，从严格有序地添加底物变为相对于ATP和泛素随机添加。通过将E1的净正向反应与E232K催化的组蛋白H2B缀合反应偶联，测定了泛素结合的内在解离常数（Kd）值。只有R54和R72残基参与游离泛素的初始结合，与野生型相比，UbR54L或UbR72L的Kd分别增加了6倍或58倍。在E1活性位点内，UbR42L和UbR72L突变体对其各自泛素腺苷酸中间体的结合表现出更显著的影响。野生型泛素腺苷酸与E1结合的估计Kd≤8×10⁻¹² M，而由UbR42L或UbR72L形成的中间体与E1的结合亲和力约低10³倍，这表明其稳定性降低≥7千卡/摩尔。（摘要截短于250字）
