Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase.
The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212-226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212-->Asp-Phe-28-->Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase.
Helmstaedt
Kerstin
K
Institut für Mikrobiologie und Genetik, Georg-August-Universität, Grisebachstrasse 8, D-37077 Göttingen, Germany.
Heinrich
Gabriele
G
Lipscomb
William N
WN
Braus
Gerhard H
GH
D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic; D001233:Aspergillus nidulans / Q000201:enzymology; D020134:Catalytic Domain; D002826:Chorismate Mutase / Q000737:chemistry / Q000378:metabolism*; D003470:Culture Media; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D024322:Amino Acids, Aromatic; D003470:Culture Media; D014443:Tyrosine; D014364:Tryptophan; D002826:Chorismate Mutase