Origin of the mutual activation of the alpha and beta 2 subunits in the alpha 2 beta 2 complex of tryptophan synthase. Effect of alanine or glycine substitutions at proline residues in the alpha subunit.
To understand how the alpha and beta 2 subunits of tryptophan synthase from Escherichia coli interact to form an alpha 2 beta 2 complex and undergo mutual activation, we have investigated alpha subunits with single amino acid replacements at conserved proline residues. Although the activities of alpha 2 beta 2 complexes that contain wild type alpha subunit or alpha subunits substituted at positions 28, 62, 96, and 207 are similar, the activities of alpha 2 beta 2 complexes that contain alpha subunits substituted at positions 57 and 132 are remarkably altered. Whereas the latter enzymes have greatly reduced activities in the individual half-reactions, they have considerably higher activities in the overall reaction. These remarkable activity results are explained by a decrease in the affinity of these mutant alpha subunits for the beta 2 subunit and by an increase in the affinity in the combined presence of ligands of both the alpha subunit and the beta 2 subunit. Isothermal calorimetric titrations of wild type beta 2 subunit with wild type alpha subunit and a mutant alpha subunit containing a substitution of glycine for proline at position 132 show that both the affinity and the exothermic association enthalpy are greatly reduced in the mutant alpha subunit although the stoichiometry of association is unchanged. The affinity of the mutant alpha subunit for the beta 2 subunits is greatly increased in the presence of an alpha subunit ligand, alpha-glycerol phosphate. We conclude that proline 132 plays a critical role in subunit interaction and in mutual subunit activation.
Ogasahara
K
K
Institute for Protein Research, Osaka University, Japan.
Hiraga
K
K
Ito
W
W
Miles
E W
EW
Yutani
K
K
D000409:Alanine; D002151:Calorimetry; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005998:Glycine; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011392:Proline; D015183:Restriction Mapping; D014367:Tryptophan Synthase / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D046911:Macromolecular Substances; D011392:Proline; D014367:Tryptophan Synthase; D000409:Alanine; D005998:Glycine