Narasimhulu S, Eddy C R, Dibartolomeis M, Kowluru R, Jefcoate C R
Biochemistry. 1985 Jul 30;24(16):4287-94. doi: 10.1021/bi00337a006.
The substrate-cytochrome P-450C-21 binding reaction has been investigated in detail by using the purified cytochrome. The apparent substrate dissociation constant (KDapp) depended on the enzyme concentration, indicating that the binding reaction does not follow simple two-component mass action equilibrium. However, the binding data fit reasonably well to a model in which the P-450C-21 exists in a monomer-dimer equilibrium and the substrate does not bind to the dimer. The intrinsic dissociation constant (K1) and the dissociation constant for the dimerization reaction (K2) were calculated from the titration data by a pattern search procedure. K1 and K2 were found to be essentially independent of the enzyme concentration, indicating the appropriateness of the assumed model. In the present study, all factors that increased the dissociation of the dimer, as indicated by an increase in K2, decreased KDapp so that it approached the intrinsic constant K1. These results suggest that there is mutual interaction of the substrate binding and self-association reactions of cytochrome P-450C-21 in the purified preparation.
利用纯化的细胞色素，已对底物 - 细胞色素P - 450C - 21结合反应进行了详细研究。表观底物解离常数（KDapp）取决于酶浓度，这表明结合反应不遵循简单的两组分质量作用平衡。然而，结合数据与一个模型相当吻合，在该模型中，P - 450C - 21以单体 - 二聚体平衡存在，且底物不与二聚体结合。通过模式搜索程序从滴定数据计算出内在解离常数（K1）和二聚化反应的解离常数（K2）。发现K1和K2基本上与酶浓度无关，这表明所假设模型的合理性。在本研究中，所有增加二聚体解离的因素，如K2的增加所示，都会降低KDapp，使其接近内在常数K1。这些结果表明，在纯化制剂中，细胞色素P - 450C - 21的底物结合和自缔合反应之间存在相互作用。
