PMID:8795043
| Citation |
Hiraga, K and Yutani, K (1996) Study of cysteine residues in the alpha subunit of Escherichia coli tryptophan synthase. 2. Role in enzymatic function. Protein Eng. 9:433-8 |
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| Abstract |
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. |
| Links | |
| Keywords |
Calorimetry; Cysteine/chemistry; Escherichia coli; Kinetics; Protein Conformation; Structure-Activity Relationship; Tryptophan Synthase/chemistry; Tryptophan Synthase/metabolism |
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