MECHANISMS OF CATALYSIS AND ALLOSTERIC REGULATION OF YEAST CHORISMATEMUTASE FROM CRYSTAL-STRUCTURES

Background: Chorismate mutase (GM) catalyzes the Claisen rearrangement of chorismate to prephenate, notably the only known enzymatically cat alyzed pericyclic reaction in primary metabolism. Structures of the en zyme in complex with an endo-oxabicyclic transition state analogue inh ibitor, previously determined for Bacillus subtilis and Escherichia co li CM, provide structural insight into the enzyme mechanism. In contra st to these bacterial CMs, yeast CM is allosterically regulated in two ways: activation by tryptophan and inhibition by tyrosine. Yeast CM e xists in two allosteric states, R (active) and T (inactive). Results: We have determined crystal structures of wild-type yeast CM cocrystall ized with tryptophan and an endo-oxabicyclic transition state analogue inhibitor, of wild-type yeast CM co-crystallized with tyrosine and th e endo-oxabicyclic transition state analogue inhibitor and of the Thr2 26-->Ser mutant of yeast CM in complex with tryptophan. Binding of the transition state analogue inhibitor to CM keeps the enzyme in a 'supe r R' state, even if the inhibitory effector tyrosine is bound to the r egulatory site. Conclusions: The endo-oxabicyclic inhibitor binds to y east CM in a similar way as it does to the distantly related CM from E . coli. The inhibitor-binding mode supports a mechanism by which polar sidechains of the enzyme bind the substrate in the pseudo-diaxial con formation, which is required for catalytic turnover. A lysine and a pr otonated glutamate sidechain have a critical role in the stabilization of the transition state of the pericyclic reaction. The allosteric tr ansition from T-->R state is accompanied by a 15 degrees rotation of o ne of the two subunits relative to the other (where 0 degrees rotation defines the T state). This rotation causes conformational changes at the dimer interface which are transmitted to the active site. An allos teric pathway is proposed to include residues Phe28, Asp24 and Glu23, which move toward the active-site cavity in the T state. In the presen ce of the transition-state analogue a super R state is formed, which i s characterised by a 22 degrees rotation of one subunit relative to th e other.