A structural view of the dissociation ofEscherichia colitryptophanase

Abstract

Tryptophanase (Trpase) is a pyridoxal 5′-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of L-tryptophan. Trpase is also known for its cold lability, which is a reversible loss of activity at low temperature (2°C) that is associated with the dissociation of the tetramer.Escherichia coliTrpase dissociates into dimers, whileProteus vulgarisTrpase dissociates into monomers. As such, this enzyme is an appropriate model to study the protein–protein interactions and quaternary structure of proteins. The aim of the present study was to understand the differences in the mode of dissociation between theE. coliandP. vulgarisTrpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. To answer this question, two groups of mutants of theE. colienzyme were created to resemble the amino-acid sequence ofP. vulgarisTrpase. In one group, residues 15 and 59 that are located along the molecular axisR(also termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axisQ(also termed the catalytic axis). Replacing amino-acid residues along theRaxis resulted in dissociation of the tetramers into monomers, similar to theP. vulgarisTrpase, while replacing amino-acid residues along theQaxis resulted in dissociation into dimers only. The crystal structure of the V59M mutant ofE. coliTrpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that inE. coliTrpase hydrophobic interactions along theRaxis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along theQaxis to a charged residue resulted in tetramers that are less susceptible to dissociation. Thus, the results indicate that dissociation ofE. coliTrpase into dimers occurs along the molecularQaxis.