Okajima T, Fukamizo T, Goto S, Fukui T, Tanizawa K
Faculty of Agriculture, Kinki University, Nara, 631-8505, Japan.
J Biochem. 1998 Aug;124(2):359-67. doi: 10.1093/oxfordjournals.jbchem.a022120.
Two types of active chimeric enzymes have been constructed by genetic engineering of chicken cytosolic adenylate kinase (AK) and porcine brain UMP/CMP kinase (UCK): one, designated as UAU, carries an AMP-binding domain of AK in the remaining body of UCK; and the other, designated as AUA, carries a UMP/CMP-binding domain of UCK in the remaining body of AK. Steady-state kinetic analysis of these chimeric enzymes revealed that UAU is 4-fold more active for AMP, 40-fold less active for UMP, and 4-fold less active for CMP than the parental UCK, although AUA has considerably lowered reactivity for both AMP and UMP. Circular dichroism spectra of the two chimeric enzymes suggest that UAU and AUA have similar folding structures to UCK and AK, respectively. Furthermore, proton NMR measurements of the UCK and UAU proteins indicate that significant differences in proton signals are limited to the aromatic region, where an imidazole C2H signal assigned to His31 shows a downfield shift upon conversion of UCK to UAU, and the signals assigned to Tyr49 and Tyr56 in the UMP/CMP-binding domain disappear in UAU. In contrast, AUA has a Tm value about 11 degreesC lower than AK, whereas UAU and UCK have similar Tm values. These results together show that the substrate specificity of nucleoside monophosphate (NMP) kinases can be engineered by the domain exchange, even though the base moiety of NMP appears to be recognized cooperatively by both the NMP-binding domain and the MgATP-binding core domain.
通过对鸡胞质腺苷酸激酶（AK）和猪脑UMP/CMP激酶（UCK）进行基因工程构建了两种活性嵌合酶：一种命名为UAU，在UCK的其余部分携带AK的AMP结合结构域；另一种命名为AUA，在AK的其余部分携带UCK的UMP/CMP结合结构域。对这些嵌合酶的稳态动力学分析表明，与亲本UCK相比，UAU对AMP的活性高4倍，对UMP的活性低40倍，对CMP的活性低4倍，尽管AUA对AMP和UMP的反应性都大大降低。两种嵌合酶的圆二色光谱表明，UAU和AUA分别具有与UCK和AK相似的折叠结构。此外，对UCK和UAU蛋白的质子核磁共振测量表明，质子信号的显著差异仅限于芳香区，在该区域，分配给His31的咪唑C2H信号在UCK转化为UAU时出现向低场移动，并且在UMP/CMP结合结构域中分配给Tyr49和Tyr56的信号在UAU中消失。相比之下，AUA的Tm值比AK低约11℃，而UAU和UCK具有相似的Tm值。这些结果共同表明，即使NMP的碱基部分似乎由NMP结合结构域和MgATP结合核心结构域协同识别，核苷单磷酸（NMP）激酶的底物特异性也可以通过结构域交换来设计。
