Han H-Y, Xu W-A, Lu Z-R, Zou F, Li S
College of Life Science, Suzhou University, Suzhou 215123, P. R. China.
J Biomol Struct Dyn. 2008 Aug;26(1):83-92. doi: 10.1080/07391102.2008.10507226.
The effect of cobalt ions (Co2+) on horseradish peroxidase (HRP) was studied in vitro by enzymatic activity assay, electronic absorption spectra, intrinsic fluorescence spectra and 8-anilo-1-naphthalenesulfonate(ANS)-binding fluorescence spectra. Co2+ at concentrations below 0.1 mM mildly increased the HRP activity, whereas higher concentrations of Co2+ significantly inactivated HRP in a time and concentration-dependent manner. Steady-state kinetic studies show that Co2+ was a noncompetitive inhibitor of o-dianisidine oxidation by HRP. The Ki value dropped as the incubation time increased. Furthermore, Co2+ was found to be an uncompetitive inhibitor of H2O2. These results suggested that Co2+ would slowly bind to the enzyme and progressively induce conformational changes. Spectroscopic analysis showed that even for high Co2+ concentrations, the structure of HRP as a whole only changed slightly; however, there were significant conformational changes near or in the active site of HRP. Based on the above results, we suggest that Co2+ may bind with some amino acids near or in the active site of HRP and the conformational changes of HRP induced by such binding should be the main reason for activation and inactivation effect of Co2+. The potential binding sites of Co2+ were also proposed.
通过酶活性测定、电子吸收光谱、内源荧光光谱和8-苯胺基-1-萘磺酸盐（ANS）结合荧光光谱等方法，在体外研究了钴离子（Co2+）对辣根过氧化物酶（HRP）的影响。浓度低于0.1 mM的Co2+能轻度提高HRP活性，而较高浓度的Co2+则以时间和浓度依赖的方式显著使HRP失活。稳态动力学研究表明，Co2+是HRP氧化邻联茴香胺的非竞争性抑制剂。随着孵育时间的增加，抑制常数（Ki）值下降。此外，发现Co2+是H2O2的反竞争性抑制剂。这些结果表明，Co2+会缓慢结合到酶上并逐渐诱导构象变化。光谱分析表明，即使对于高浓度的Co2+，HRP的整体结构仅略有变化；然而，在HRP活性位点附近或活性位点处存在显著的构象变化。基于上述结果，我们认为Co2+可能与HRP活性位点附近或活性位点处的一些氨基酸结合，并且这种结合诱导的HRP构象变化应该是Co2+激活和失活作用的主要原因。还提出了Co2+的潜在结合位点。
