Monofunctional Chorismate Mutase from Bacillus subtilis: FTIR Studies and the Mechanism of Action of the Enzyme

Abstract

The Fourier transform infrared (FTIR) spectrum of the complex between prephenate and the monofunctional chorismate mutase from Bacillus subtilis displays one prominent band at 1714 cm-1. Using isotopically-labeled ligand, we have shown that this band corresponds to the ketonic carbonyl stretching vibration of enzyme-bound prephenate. The frequency of this carbonyl vibration of prephenate does not change significantly on binding to the protein. These data indicate that chorismate mutase does not use electrophilic catalysis in the rearrangement of chorismate. A comparison of the resolution-enhanced FTIR spectra of the unliganded mutase and of the protein complexed with its ligands reveals marked differences in the amide I' vibration band. These changes suggest that structural alterations in the protein occur upon binding prephenate. When combined with information from the crystal structure of the enzyme and its complexes, it appears that significant ordering of the C-terminal region occurs upon ligand binding. These changes at the active site may be important for efficient catalysis and likely influence the association and dissociation rates of the enzyme and its ligands. The enzymic rearrangement of chorismate evidently proceeds via a pericyclic process, and much, if not all, of the rate acceleration derives from the selective binding of the appropriate conformer of the substrate, with some additional contribution possible from electrostatic stabilization of the transition state.

The mechanism of catalysis of the chorismate to prephenate reaction by the Escherichia coli mutase enzyme

“…From the highly polar TS structure and the electrostatic interactions observed in the crystal structure of EcCM complexed with the TSA, it has been proposed that tighter binding of TS compared with substrate (CHOR) by EcCM is responsible for the Ͼ10 6 catalytic enhancement (8,20,(25)(26)(27). The 100-fold tighter binding (K i ͞K m ϭ 100) of TSA compared with CHOR has been interpreted as being caused by TSA being a transition state analogue.…”

Location of the active site of allosteric chorismate mutase from Saccharomyces cerevisiae, and comments on the catalytic and regulatory mechanisms.

“…Furthermore, the very low sequence similarity observed among chorismate mutases from different sources provides more alternatives and advantages in dissecting the catalytic pathways. Until now it has been generally accepted that catalysis proceeds via a chair-like transition state (3-6) and that the enzyme accelerates the reaction by selecting the active conformer of the substrate and by stabilizing the transition state in an energetically favorable environment of the active site without the involvement of any functional group (7,8).…”

“…Furthermore, the very low sequence similarity observed among chorismate mutases from different sources provides more alternatives and advantages in dissecting the catalytic pathways. Until now it has been generally accepted that catalysis proceeds via a chair-like transition state (3-6) and that the enzyme accelerates the reaction by selecting the active conformer of the substrate and by stabilizing the transition state in an energetically favorable environment of the active site without the involvement of any functional group (7,8).The recently determined structures of chorismate mutases from Bacillus subtilis (BCM) (7), yeast Saccharomyces cerevisiae (YCM) (9), and Escherichia coli (ECM) (10, 11) provide insights into the catalytic mechanism. Among these three enzymes, the YCM is unique in that its activity is regulated by an allosteric mechanism in which tryptophan is an activator and tyrosine is an inhibitor (12).…”

A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions

“…The data indicate that both the uncatalyzed and catalyzed reactions proceed through a transition state with chairlike geometry. Different models for the mechanism of the enzymatic catalysis have been suggested, including nucleophilic attack of an active side residue or conformational restriction of the substrate by binding to the active site (9,10). In solution 10-20% of chorismate exists as the energetically less favored pseudodiaxial form in dynamic equilibrium with the pseudodiequatorial form (11).…”

“…It is assumed that the reaction is accelerated by binding of this active conformer to the enzyme, which locks the substrate via a series of electrostatic and hydrogen bonding and van der Waals interactions in the requisite chair conformation for rearrangement (12)(13)(14)(15)(16). Furthermore, the transition state might be stabilized by electrostatic interactions with active site residues (10,17). In an alternative mechanism an acidic residue is proposed to protonate the ether oxygen O7 of the substrate chorismate (9).…”