Arribas J, Castaño J G
Departmento de Bioquímica e Instituto de Investigaciones Biomédicas del Consejo Superior de Investigaciones Científicas, Facultad de Medicina de la UAM, Madrid, Spain.
J Biol Chem. 1990 Aug 15;265(23):13969-73.
We present here a detailed study of the effect of detergents on the three peptidase activities (hydrolysis of the LLVY, ARR, and LLE peptides) of the purified multicatalytic proteinase from rat liver. At Triton X-100 and sodium dodecyl sulfate (SDS) concentrations of 0.1%, all three peptidase activities are inhibited. Lower concentrations of the two detergents (0.01%) do not affect the hydrolysis of the ARR peptide, whereas they behave differently on the hydrolysis of the LLVY and LLE peptides. Triton X-100 inhibits and SDS strongly activates LLVY peptide hydrolysis by decreasing and increasing Vmax, respectively. In the absence of detergents, the saturation curve for the LLE peptide can be analyzed as the result of two components, one showing cooperative (nH = 1.6) with higher affinity (S0.5 = 60 microM) and lower Vmax than a second, noncooperative component (Km = 320 microM). SDS (0.01%) activates LLE peptide hydrolysis by suppressing cooperativity, slightly increasing Vmax, and decreasing the half-saturation concentration (Km = 30 microM) of the enzyme. Triton X-100 (0.01%) also suppresses the cooperativity and decreases the half-saturation concentration (Km = 25 microM) for the LLE peptide; in contrast, it reduces Vmax by inhibition of the low affinity, high Vmax component observed in the absence of detergents. Based on these observations, it can be concluded that both detergents behave like allosteric activators of peptidylglutamyl-peptide hydrolyzing activity and that the multicatalytic proteinase has at least three different classes of active sites: two independent noncooperative sites that catalyze the hydrolysis of trypsin and chymotrypsin-like substrates and one class for peptidylglutamyl-peptide hydrolysis having two components: one cooperative (two or more sites) and one noncooperative.
我们在此展示了一项关于去污剂对从大鼠肝脏中纯化得到的多催化蛋白酶的三种肽酶活性（LLVY、ARR和LLE肽的水解）影响的详细研究。在Triton X-100和十二烷基硫酸钠（SDS）浓度为0.1%时，所有三种肽酶活性均受到抑制。两种去污剂的较低浓度（0.01%）不影响ARR肽的水解，而它们对LLVY和LLE肽的水解表现不同。Triton X-100通过分别降低和增加Vmax来抑制和强烈激活LLVY肽的水解。在没有去污剂的情况下，LLE肽的饱和曲线可分析为两个组分的结果，一个显示出协同作用（nH = 1.6），与第二个非协同组分相比具有更高的亲和力（S0.5 = 60 microM）和更低的Vmax（Km = 320 microM）。SDS（0.01%）通过抑制协同作用、略微增加Vmax以及降低酶的半饱和浓度（Km = 30 microM）来激活LLE肽的水解。Triton X-100（0.01%）也抑制协同作用并降低LLE肽的半饱和浓度（Km = 25 microM）；相反，它通过抑制在没有去污剂时观察到的低亲和力、高Vmax组分来降低Vmax。基于这些观察结果，可以得出结论，两种去污剂都表现为肽基谷氨酰肽水解活性的别构激活剂，并且多催化蛋白酶至少有三类不同的活性位点：两个独立的非协同位点，催化胰蛋白酶和类胰凝乳蛋白酶底物的水解，以及一类用于肽基谷氨酰肽水解的位点，具有两个组分：一个协同（两个或更多位点）和一个非协同。
