Roberts Tania M, Merrill A Rod
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Biochem J. 2002 Nov 1;367(Pt 3):601-8. doi: 10.1042/BJ20020768.
CRM66 (cross-reactive 66 kDa protein) is an inactive mutant form of Pseudomonas aeruginosa exotoxin A that has been isolated from a mutant strain of P. aeruginosa derived from nitrosoguanidine-based mutagenesis. The mutation within this enzyme toxin was previously identified as H426Y and it was shown to possess significantly reduced enzymic activity. Furthermore, it was previously suggested that His-426 may directly participate in the catalytic mechanism of the exotoxin A enzyme and that it may also play an important role in the binding of the protein substrate of exotoxin A, a critical protein factor in eukaryotic protein translation known as elongation factor-2. In order to more thoroughly characterize the role of His-426 in the enzyme mechanism of exotoxin A, amino acid substitutions were made within helix 1 of the enzyme domain in the vicinity of the His-426 residue. Analysis of the site-directed mutagenesis results involving kinetic and protein structural integrity measurements revealed that His-426 H-bonds to Tyr-502 and that replacement of His-426 with polar substitutions leads to structural alterations of the enzyme's folded conformation. Furthermore, it was shown that His-426 is not important for the binding of either of the two substrates of exotoxin A, NAD(+) or elongation factor-2. In summary, these data show that His-426 is not an active-site residue and that it is not important for substrate binding or orientation, but that it plays an important structural role in helping to maintain the folded conformation of the enzyme toxin. Therefore, the role of His-426 would seem to be to tether helix 1 to the main body of the enzyme, and mutations resulting in the disruption of this region of the enzyme result in a significantly impaired enzyme.
CRM66（交叉反应性66 kDa蛋白）是铜绿假单胞菌外毒素A的一种无活性突变形式，它是从基于亚硝基胍诱变产生的铜绿假单胞菌突变株中分离出来的。这种酶毒素中的突变先前被鉴定为H426Y，并且显示其酶活性显著降低。此外，先前有人提出，组氨酸-426可能直接参与外毒素A酶的催化机制，并且它可能在外毒素A的蛋白质底物（一种在真核生物蛋白质翻译中起关键作用的蛋白质因子，称为延伸因子-2）的结合中也发挥重要作用。为了更全面地描述组氨酸-426在外毒素A酶机制中的作用，在组氨酸-426残基附近的酶结构域的螺旋1内进行了氨基酸替换。对涉及动力学和蛋白质结构完整性测量的定点诱变结果的分析表明，组氨酸-426与酪氨酸-502形成氢键，并且用极性替换物取代组氨酸-426会导致酶折叠构象的结构改变。此外，研究表明，组氨酸-426对外毒素A的两种底物NAD(+)或延伸因子-2的结合并不重要。总之，这些数据表明，组氨酸-426不是活性位点残基，对底物结合或定向也不重要，但它在帮助维持酶毒素的折叠构象方面发挥重要的结构作用。因此，组氨酸-426的作用似乎是将螺旋1与酶的主体相连，导致该酶这一区域被破坏的突变会导致酶显著受损。
