Carvajal N, Torres C, Uribe E, Salas M
Departamento de Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Chile.
Comp Biochem Physiol B Biochem Mol Biol. 1995 Sep;112(1):153-9. doi: 10.1016/0305-0491(95)00027-6.
As determined by atomic absorption, fully activated human liver arginase contained 1.1 +/- 0.1 Mn2+/subunit. Upon dissociation to inactive subunits (< 0.01 Mn2+/subunit), there was decreased intensity and a red shift in the tryptophan fluorescence emission spectra of the enzyme, and the resulting species were markedly sensitive to thermal and proteolytic inactivation by trypsin. Arginine and lysine specifically protected the subunits from heat inactivation. Subunit activation by Mn2+ followed hyperbolic kinetics (Kd = 0.08 +/- 0.01 microM). In addition to Mn2+, Ni2+ and Co2+ converted inactive subunits into active monomers, and favoured their association to the oligomeric state of the enzyme (M(r) = 120,000 +/- 2000). The replacement of Mn2+ by Ni2+ or Co2+ resulted in significant changes in Vmax without any change in the Km values for the substrates (arginine or canavanine) or the Ki value for lysine inhibition. The results support our previous suggestion (Carvajal et al., 1994) that Mn2+ is not essential for substrate binding to arginase, and substantiates the conclusion that species differences may exist in the interaction of arginase with metal ions.
通过原子吸收测定，完全活化的人肝脏精氨酸酶每个亚基含有1.1±0.1个Mn2+。当解离为无活性亚基时（每个亚基<0.01个Mn2+），该酶的色氨酸荧光发射光谱强度降低且发生红移，并且所得物种对胰蛋白酶的热失活和蛋白水解失活极为敏感。精氨酸和赖氨酸可特异性保护亚基免受热失活。Mn2+介导的亚基活化遵循双曲线动力学（Kd = 0.08±0.01 microM）。除了Mn2+，Ni2+和Co2+也可将无活性亚基转化为活性单体，并促进它们缔合形成酶的寡聚状态（M(r)= 120,000±2000）。用Ni2+或Co2+替代Mn2+会导致Vmax发生显著变化，而底物（精氨酸或刀豆氨酸）的Km值或赖氨酸抑制的Ki值均无变化。这些结果支持了我们之前的推测（Carvajal等人，1994年），即Mn2+对于底物与精氨酸酶的结合并非必不可少，并且证实了精氨酸酶与金属离子相互作用可能存在物种差异的结论。
