Subcellular Biochemistry, Proteins: Structure, Function and Protein Engineering (Biswas
- E. W. Miles
The interaction of the a subunit with the b2 subunit of tryptophan synthase is known to be necessary for the activation of each subunit and for the catalytic efficiency of the a2b2 complex. To elucidate the roles of hydrogen bonds in the interaction site between the a and b subunits for subunit association, eight mutant a subunits at five hydrogen bonding residues (N104D, N104A, N108D, N108A, E134A, E135A, N157D, and N157A) were constructed, and the thermodynamic parameters of association with the b subunit were obtained using a titration calorimeter. The N104D and N104A mutations remarkably decreased the stimulation activities, the association constants, and the association enthalpies. Although the association constant and the stimulation activities of E134A were reduced in the absence of salt, the change in the association enthalpy was relatively small, and the addition of salt could repair its defects. The substitutions at positions 135 and 157 did not affect the stimulation activity and decreased the Gibbs energy of association corresponding to the defect in 1 mol of hydrogen bond. The present results suggest that the a subunit which has amutation at position 104 cannot fold into an intact conformation upon complex formation, resulting in reduced stimulation activities. The hydrogen bond with Asn-104, which is a conserved residue among 16 microorganisms, was especially important for a/b interaction and mutual activation.