Yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution

Abstract

Chorismate mutase, a branch-point enzyme in the aromatic amino acid pathway of Saccharomyces cerevisiae, and also a mutant chorismate mutase with a single amino acid substitution in the C-terminal part of the protein have been purified approximately 20-fold and 64-fold from overproducing strains, respectively. The wild-type enzyme is activated by tryptophan and subject to feedback inhibition by tyrosine, whereas the mutant enzyme does not respond to activation by tryptophan nor inhibition by tyrosine. Both enzymes are dimers consisting of two identical subunits of Mr 30,000, each one capable of binding one substrate and one activator molecule. Each subunit of the wild-type enzyme also binds one inhibitor molecule, whereas the mutant enzyme lost this ability. The enzyme reaction was observed by 1H NMR and shows a direct and irreversible conversion of chorismate to prephenate without the accumulation of any enzyme-free intermediates. The kinetic data of the wild-type chorismate mutase show positive cooperativity toward the substrate with a Hill coefficient of 1.71 and a [S]0.5 value of 4.0 mM. In the presence of the activator tryptophan, the cooperativity is lost. The enzyme has an [S]0.5 value of 1.2 mM in the presence of 10 microM tryptophan and an increased [S]0.5 value of 8.6 mM in the presence of 300 microM tyrosine. In the mutant enzyme, a loss of cooperativity was observed, and [S]0.5 was reduced to 1.0 mM. This enzyme is therefore locked in the activated state by a single amino acid substitution.

Evolution of 3-deoxy- d - arabino -heptulosonate-7-phosphate synthase-encoding genes in the yeast Saccharomyces cerevisiae

“…At intracellular Trp levels of 20 M, at which anthranilate synthase is not yet strongly inhibited by Trp (K i 56 M), this amino acid already activates chorismate mutase (K a , 1.5 M) leading to increased Phe and Tyr synthesis. When Tyr and Phe are being synthesized, the DAHPSs at the beginning of the pathway (K i , 10 M and 1 M) as well as chorismate mutase (K i , 50 M) are inhibited, making an additional Trp control dispensable (19,20,41,52).…”

Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase

“…Perhaps, on the other hand, these rearrangements upon monomerization cause reduction of activity by reorientation of crucial residues of the active site. On the other hand, it cannot yet be excluded that the substitutions introduced at positions 28 and 212 account for the change in activity, because single substitutions in loop L220s can strongly affect enzyme activity in some cases (5,26). However, because the pH optimum of the monomer equaled that of the unliganded wt enzyme, conformational rearrangements cannot be very pronounced.…”

“…In addition, residues from loop L50s and helix H4 from one subunit interact with loop L80s from the other subunit, leading to formation of another two hydrophobic cores. These interactions were so strong that dissociation of the dimers occurred only after addition of 4 M guanidine hydrochloride upon unfolding of the monomers (5).…”

Coevolution of transcriptional and allosteric regulation at the chorismate metabolic branch point of Saccharomyces cerevisiae

“…The yeast ARO7 c allele encoding an unregulated CM displays high catalytic activity that preferentially channels chorismate toward the tyrosine͞ phenylalanine branch when expressed in high amounts (11). To modulate distribution of chorismate, we replaced the wild-type ARO7 gene in strain RH1408 [gcn4-103, ura3-52] at its original locus with the ARO7 c allele, resulting in strain RH2463.…”

“…Tyrosine inhibits activity by decreasing the affinity of the enzyme toward its substrate, whereas tryptophan, the end product of the opposite branch, acts as a strong activator of catalytic turnover, resulting in Michaelis-Menten kinetics without cooperativity. Expression of the ARO7 T226I mutant allele, here referred to as ARO7 c , conserves the latter situation and leads to an unregulated, noncooperative yeast CM that is locked in its allosteric R state (11). In contrast to most amino acid biosynthetic genes, the CM-encoding ARO7 gene of S. cerevisiae is not transcriptionally regulated by the general control activator Gcn4p (12).…”