Hiraga K, Yutani K
Institute for Protein Research, Osaka University, Japan.
Protein Eng. 1996 May;9(5):433-8. doi: 10.1093/protein/9.5.433.
To understand the functional roles of Cys residues in the alpha subunit of tryptophan synthase from Escherichia coli, single mutants of the alpha subunit, in which each of the three Cys residues was substituted with Ser, Gly, Ala or Val, were constructed by site-directed mutagenesis. The effects of the substitutions on the function of tryptophan synthase were investigated by activity measurements, calorimetric measurements of association with the beta subunit and steady-state kinetic analysis of catalysis. Although the three Cys residues are located away from the apparently important parts for enzymatic activity, substitutions at position 81 by Ser, Ala or Val caused decreases in the intrinsic activity of the alpha subunit. Furthermore, Cys81Ser and Cys81Val reduced stimulation activities in the alpha and beta reactions due to formation of a complex with the beta subunit. The lower stimulation activities of the mutant proteins were not correlated with their abilities to associate with the beta 2 subunit but were correlated with decreases in kcat. The present results suggest that position 81 plays an indirectly important role in the activity of the alpha subunit itself and the mutual activation mechanism of the complex.
为了了解大肠杆菌色氨酸合酶α亚基中半胱氨酸残基的功能作用，通过定点诱变构建了α亚基的单突变体，其中三个半胱氨酸残基分别被丝氨酸、甘氨酸、丙氨酸或缬氨酸取代。通过活性测量、与β亚基结合的量热测量以及催化的稳态动力学分析，研究了这些取代对色氨酸合酶功能的影响。尽管这三个半胱氨酸残基位于远离酶活性明显重要部分的位置，但81位被丝氨酸、丙氨酸或缬氨酸取代会导致α亚基内在活性降低。此外，Cys81Ser和Cys81Val由于与β亚基形成复合物，降低了α和β反应中的刺激活性。突变蛋白较低的刺激活性与其与β2亚基结合的能力无关，而是与kcat的降低有关。目前的结果表明，81位在α亚基本身的活性和复合物的相互激活机制中起着间接重要的作用。
