Schlattner U, Eder M, Dolder M, Khuchua Z A, Strauss A W, Wallimann T
Institute of Cell Biology, Swiss Federal Institute of Technology (ETH), Hönggerberg, Zurich.
Biol Chem. 2000 Nov;381(11):1063-70. doi: 10.1515/BC.2000.131.
The mitochondrial isoenzymes of creatine kinase (MtCK), ubiquitous uMtCK and sarcomeric sMtCK, are key enzymes of oxidative cellular energy metabolism and play an important role in human health and disease. Very little is known about uMtCK in general, or about sMtCK of human origin. Here we have heterologously expressed and purified both human MtCK isoenzymes to perform a biochemical, kinetic and structural characterization. Both isoenzymes occurred as octamers, which can dissociate into dimers. Distinct Stokes' radii of uMtCK and sMtCK in solution were indicative for conformational differences between these equally sized proteins. Both human MtCKs formed 2D-crystals on cardiolipin layers, which revealed further subtle differences in octamer structure and stability. Octameric human sMtCK displayed p4 symmetry with lattice parameters of 145 A, indicating a 'flattening' of the octamer on the phospholipid layer. pH optima and enzyme kinetic constants of the two human isoenzymes were significantly different. A pronounced substrate binding synergism (Kd > Km) was observed for all substrates, but was most pronounced in the forward reaction (PCr production) of uMtCK and led to a significantly lower Km for creatine (1.01 mM) and ATP (0.11 mM) as compared to sMtCK (creatine, 7.31 mM; ATP, 0.68 mM).
肌酸激酶的线粒体同工酶（MtCK），即普遍存在的uMtCK和肌节sMtCK，是细胞氧化能量代谢的关键酶，在人类健康和疾病中发挥着重要作用。一般来说，人们对uMtCK了解甚少，对源自人类的sMtCK也知之甚少。在此，我们通过异源表达和纯化两种人类MtCK同工酶，对其进行生化、动力学和结构表征。两种同工酶均以八聚体形式存在，可解离为二聚体。溶液中uMtCK和sMtCK不同的斯托克斯半径表明这些大小相同的蛋白质之间存在构象差异。两种人类MtCK均在心磷脂层上形成二维晶体，这揭示了八聚体结构和稳定性方面更细微的差异。八聚体人类sMtCK呈现p4对称性，晶格参数为145 Å，表明八聚体在磷脂层上“扁平化”。两种人类同工酶的最适pH值和酶动力学常数存在显著差异。所有底物均观察到明显的底物结合协同作用（Kd > Km），但在uMtCK的正向反应（磷酸肌酸生成）中最为明显，与sMtCK（肌酸，7.31 mM；ATP，0.68 mM）相比，导致肌酸（1.01 mM）和ATP（0.11 mM）的Km显著降低。
