Muratore Kathryn E, Seeliger Markus A, Wang Zhihong, Fomina Dina, Neiswinger Johnathan, Havranek James J, Baker David, Kuriyan John, Cole Philip A
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Biochemistry. 2009 Apr 21;48(15):3378-86. doi: 10.1021/bi900057g.
Protein tyrosine kinases are critical cell signaling enzymes. These enzymes have a highly conserved Arg residue in their catalytic loop which is present two residues or four residues downstream from an absolutely conserved Asp catalytic base. Prior studies on protein tyrosine kinases Csk and Src revealed the potential for chemical rescue of catalytically deficient mutant kinases (Arg to Ala mutations) by small diamino compounds, particularly imidazole; however, the potency and efficiency of rescue was greater for Src. This current study further examines the structural and kinetic basis of rescue for mutant Src as compared to mutant Abl tyrosine kinase. An X-ray crystal structure of R388A Src revealed the surprising finding that a histidine residue of the N-terminus of a symmetry-related kinase inserts into the active site of the adjacent Src and mimics the hydrogen-bonding pattern seen in wild-type protein tyrosine kinases. Abl R367A shows potent and efficient rescue more comparable to Src, even though its catalytic loop is more like that of Csk. Various enzyme redesigns of the active sites indicate that the degree and specificity of rescue are somewhat flexible, but the overall properties of the enzymes and rescue agents play an overarching role. The newly discovered rescue agent 2-aminoimidazole is about as efficient as imidazole in rescuing R/A Src and Abl. Rate vs pH studies with these imidazole analogues suggest that the protonated imidazolium is the preferred form for chemical rescue, consistent with structural models. The efficient rescue seen with mutant Abl points to the potential of this approach to be used effectively to analyze Abl phosphorylation pathways in cells.
蛋白酪氨酸激酶是关键的细胞信号酶。这些酶在其催化环中具有一个高度保守的精氨酸残基，该残基位于绝对保守的天冬氨酸催化碱基下游两个残基或四个残基处。先前对蛋白酪氨酸激酶Csk和Src的研究表明，小的二氨基化合物，特别是咪唑，有可能对催化缺陷型突变激酶（精氨酸突变为丙氨酸）进行化学拯救；然而，Src的拯救效力和效率更高。本研究进一步探讨了与突变型Abl酪氨酸激酶相比，突变型Src拯救的结构和动力学基础。R388A Src的X射线晶体结构揭示了一个惊人的发现，即对称相关激酶N端的一个组氨酸残基插入相邻Src的活性位点，并模拟野生型蛋白酪氨酸激酶中的氢键模式。Abl R367A显示出与Src更相似的高效拯救，尽管其催化环更类似于Csk。对活性位点的各种酶重新设计表明，拯救的程度和特异性有些灵活，但酶和拯救剂的总体性质起着主导作用。新发现的拯救剂2-氨基咪唑在拯救R/A Src和Abl方面与咪唑效率相当。对这些咪唑类似物的速率与pH研究表明，质子化的咪唑鎓是化学拯救的首选形式，这与结构模型一致。突变型Abl的高效拯救表明这种方法有可能有效地用于分析细胞中的Abl磷酸化途径。
