Brown Kenneth L, Zou Xiang, Chen Guodong, Xia Zuping, Marques Helder M
Department of Chemistry and Biochemistry, Clippinger Laboratories, Ohio University, Athens, OH 45701, USA.
J Inorg Biochem. 2004 Feb;98(2):287-300. doi: 10.1016/j.jinorgbio.2003.10.016.
The coenzymic activity of eight analogs of coenzyme B(12) (5'-deoxyadenosyl-cobalamin, AdoCbl) with structural alterations in the Ado ligand has been investigated with the AdoCbl-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii. Six of the analogs were partially active coenzymes, and one, 3-iso-5'-deoxyadenosylcobalamin (3-IsoAdoCbl) was nearly as active as AdoCbl itself. NMR-restrained molecular modeling of 3-IsoAdoCbl revealed a highly conformationally mobile structure which required a four state model to be consistent with the NMR data. Thus, two conformations, one with the IsoAdo ligand over the eastern quadrant of the corrin, and one with the IsoAdo ligand over the northern quadrant, each undergo a facile syn/anti conformational equilibrium in the IsoAdo ligand. Spectrophotometric measurement of the kinetics of RTPR-induced cleavage of the carbon-cobalt bond of 3-IsoAdoCbl showed that it binds to the enzyme with the same affinity as AdoCbl, but its homolysis is only 20% as rapid. Investigation of the non-enzymatic thermolysis of 3-IsoAdoCbl showed that like AdoCbl, 3-IsoAdoCbl decomposes by competing homolytic and heterolytic pathways. A complete temperature-dependent kinetic and product analysis, followed by correction for the base-off species permitted deconvolution of the specific rate constant for both pathways. Eyring plots for the homolysis and heterolysis rate constant cross at 93 degrees C, so that homolysis is the predominant pathway at high temperature, but heterolysis is the predominant pathway at low temperature. At 37 degrees C, the homolysis of 3-IsoAdoCbl is 5.5-fold faster than that of AdoCbl, and the enzyme catalyzes carbon-cobalt bond homolysis in 3-IsoAdoCbl by a factor of 5.9 x 10(7), only 3.9% of the catalytic efficiency with AdoCbl itself. It seems likely that the conformational flexibility of 3-IsoAdoCbl allows it to adopt a coformation in which the hydrogen bonding patterns of the adenine moiety are similar to those of AdoCbl itself, and that this is responsible for the high enzymatic activity of this analog.
利用来自莱氏乳杆菌的依赖于辅酶B12（5'-脱氧腺苷钴胺素，AdoCbl）的核糖核苷三磷酸还原酶（RTPR），研究了Ado配体结构改变的8种辅酶B12类似物的辅酶活性。其中6种类似物是部分活性辅酶，而一种3-异-5'-脱氧腺苷钴胺素（3-IsoAdoCbl）的活性几乎与AdoCbl本身相同。对3-IsoAdoCbl进行的核磁共振约束分子建模显示，其结构具有高度的构象流动性，需要一个四态模型才能与核磁共振数据一致。因此，两种构象，一种是IsoAdo配体位于咕啉的东象限上方，另一种是IsoAdo配体位于北象限上方，每种构象在IsoAdo配体中都经历了容易的顺式/反式构象平衡。对RTPR诱导的3-IsoAdoCbl碳钴键断裂动力学进行分光光度测量表明，它与酶的结合亲和力与AdoCbl相同，但其均裂速度仅为AdoCbl的20%。对3-IsoAdoCbl的非酶热解研究表明，与AdoCbl一样，3-IsoAdoCbl通过竞争均裂和异裂途径分解。进行完整的温度依赖性动力学和产物分析，然后对碱基脱离物种进行校正，从而可以解卷积这两种途径的比速率常数。均裂和异裂速率常数的艾林曲线在93℃相交，因此在高温下均裂是主要途径，而在低温下异裂是主要途径。在37℃时，3-IsoAdoCbl的均裂速度比AdoCbl快5.5倍，并且该酶催化3-IsoAdoCbl中碳钴键均裂的效率是AdoCbl本身的5.9×10⁷倍，仅为AdoCbl催化效率的3.9%。3-IsoAdoCbl的构象灵活性似乎使其能够采取一种构象，其中腺嘌呤部分的氢键模式与AdoCbl本身的相似，并且这就是该类似物具有高酶活性的原因。
