Mutations in the contact region between the alpha and beta subunits of tryptophan synthase alter subunit interaction and intersubunit communication.
Interaction between the alpha and beta subunits of tryptophan synthase leads to mutual stabilization of the active conformations and to coordinated control of the activities of the two subunits. To elucidate the roles of specific residues in the interaction site between the alpha and beta subunits, mutant alpha and beta subunits were constructed, and the effects of mutation on subunit interaction and intersubunit communication were determined. Mutation of either alpha subunit Asp56 (alphaD56A) or beta subunit Lys167 (betaK167T), residues that interact in some crystal structures of the tryptophan synthase alpha2beta2 complex, decreases the ability of the alpha subunit to activate the beta subunit and alters the reaction and substrate specificity of the beta subunit. Partial conformational repair is provided by alpha-glycerol 3-phosphate, a ligand that binds to the alpha subunit, or by Cs+ or NH4+, ligands that bind to the beta subunit. Mutation of beta subunit Arg175 (betaR175A), a residue that interacts with alpha subunit Pro57 in some structures, has much smaller effects on activity but results in a 15-fold increase in the apparent Kd for dissociation of the alpha and beta subunits. Replacement of the single tryptophan in the beta subunit by phenylalanine (W177F) has only small effects on activity but increases the apparent subunit dissociation constant approximately 10-fold. The most important conclusions of this investigation are that interaction between alphaAsp56 and betaLys167 is important for intersubunit communication and that mutual stabilization of the active conformations of the two subunits is impaired by mutation of either residue.
Rowlett
R
R
Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Yang
L H
LH
Ahmed
S A
SA
McPhie
P
P
Jhee
K H
KH
Miles
E W
EW
D019943:Amino Acid Substitution; D001483:Base Sequence; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics*
D008024:Ligands; D014367:Tryptophan Synthase