Krishnamoorthy G, Prabhananda B S
Biochim Biophys Acta. 1982 Dec 20;709(2):234-46. doi: 10.1016/0167-4838(82)90466-6.
Difference spectra have shown that the dissociation constant associated with the dominant species, formed by the binding of bromophenol blue or bromocresol purple to lysozyme, is not sensitive to pH in the range 6-9.5. This was confirmed from temperature-jump studies. However, the inhibition of lysozyme catalysed cell lysis by these dyes is dependent on pH and ionic strength. In the reaction scheme, which takes note of both these observations, we have to consider the formation of an enzyme-dye substrate complex (EDS) which has the same kcat as does the enzyme-substrate complex (ES). The formation of EDS from ES and free dye (D) is controlled by an ionisable group. Analysis of the data using an equation similar to that of Maurel and Douzou gives a pK of approx. 5.6 for this group and this pK is close to that of histidine-15. The inhibition mainly comes from the difference in the formation constants of EDS and ES. The initial binding site of the substrate (S) in ES is not in the cleft region A-F. The cell lysis takes place after S binds in the cleft, in a subsequent step. The rate constants of this step are included in kcat. (kcat is obtained by analysing activity using the simple Michaelis-Menten kinetics). Inhibition by chitotriose also supports this conclusion. The dye binding site is also suggested to be close to histidine-15. Experimental results support the contention that the electrostatic potential due to the negatively charged cell wall substrate could alter the effective pK of ionisable groups on the enzyme in ES.
差示光谱表明，由溴酚蓝或溴甲酚紫与溶菌酶结合形成的主要物种的解离常数在6 - 9.5范围内对pH不敏感。这一点通过温度跃变研究得到了证实。然而，这些染料对溶菌酶催化的细胞裂解的抑制作用取决于pH和离子强度。在考虑到这两个观察结果的反应方案中，我们必须考虑形成一种酶 - 染料底物复合物（EDS），它与酶 - 底物复合物（ES）具有相同的催化常数（kcat）。从ES和游离染料（D）形成EDS受一个可电离基团控制。使用类似于Maurel和Douzou的方程对数据进行分析，得出该基团的pK约为5.6，且这个pK接近组氨酸 - 15的pK。抑制作用主要源于EDS和ES形成常数的差异。底物（S）在ES中的初始结合位点不在裂隙区域A - F中。细胞裂解在S结合到裂隙后，在随后的步骤中发生。该步骤的速率常数包含在kcat中。（kcat是通过使用简单的米氏动力学分析活性获得的）。壳三糖的抑制作用也支持这一结论。染料结合位点也被认为靠近组氨酸 - 15。实验结果支持这样的观点，即带负电荷的细胞壁底物产生的静电势可能会改变ES中酶上可电离基团的有效pK。
