Chorismate mutase, inhibition

The conversion of [49] into [50] involves a Claisen rearrangement. Once this was realized it was less surprising that no specific catalytic groups on the enzyme are involved. Support for the Claisen-type mechanism comes from the inhibition shown by the bicyclic dicarboxylate [51], prepared by Bartlett and Johnson (1985) as an analogue of the presumed transition state [52], This same structure [51], coupled through the hydroxyl group to a small protein, was used as a hapten to induce antibodies, one (out of eight) of which mimics the behaviour of chorismate mutase, albeit less efficiently (Table 7). 

Although chorismate mutase does provide a rate enhancement of 2 X 10 (147), this uni-molecular reaction readily occurs without enzyme, under mild conditions. The reaction was expected to pass through a chairlike transition state (59)(Fig. 17.25) but early molecular orbital calculations indicated that the boatlike transition state (60) was not out of the question (147). In an attempt to define the transition-state structure, several compounds, each designed to mimic a putative transition state, were synthesized and tested as chorismate mutase inhibitors (147). The enzyme was found to be inhibited by the exo-carboxy nonane (61), with an apparent value of 3.9 X 10 M Conversely, the endo-carboxy nonane (62) did not inhibit the enzyme. The apparent K- value of the adaman-... 

The largest flux of carbon atoms from chorismate goes into the phenylala-nine/tyrosine pathway, among others leading to lignin and important groups of secondary metabolites such as flavonoids and anthocyanins. The first enzyme in that particular pathway, chorismate mutase (CM, EC 5.4.99.5), catalyzes the conversion of chorismate to prephenate (Fig. 8). Both a cytosolic and a plastidial form have been detected in several plants (e.g., 144-147). The plastidial isoform is inhibited by phenylalanine and tyrosine, and activated by tryptophan the other isoform is not affected by these... 

Figure 4. Enzymes of Rhizobium (a) and Lemna (b) proposed as sites of glyphosate inhibition of aromatic amino acid synthesis. Abbreviations CM, chorismate mutase PDH, prephenate dehydrogenase and PD, prephenate dehydratase.

Studies with isolated enzymes in vitro reveal feedback inhibition of chorismate mutase by phenylalanine and tyrosine. Tryptophan apparently controls its own synthesis by feedback inhibition of anthranilate synthase and furthermore exerts control in the partitioning of chorismate between the two competing routes of chorismate metabolism by its ability to both activate chorismate mutase and relieve the inhibition imposed on this step by phenylalanine and tyrosine. In addition, carbon flux throuch chorismate to prephenate is also sensitive to fluctuations in chorismate concentration due to the allosteric substrate activation of chorismate mutase by chorismate. 

Fig. 6. Regulation of the synthesis of aromatic amino acids in plants. Each of the sequential arrows represents the enzyme catalyzed steps detected in Figs. 2, 3 and 4. Bold lines indicate inhibition of chorismate mutase by phenylalanine and tyrosine in addition to anthranilate synthase inhibition by tryptophan. The dashed arrow symbolizes the ability of tryptophan to both activate chorismate mutase and antagonize inhibition of this enzyme by either phenylalanine or tyrosine.

In higher plants, phenylalanine seems to be formed in an alternative manner by formation of prephenate (26) and conversion of this intermediate into arogenic acid (32). Chorismate mutase occurs as two isozymes which have been purified to homogeneity from mung bean and sorghum. All chorismate mutase isozymes show allosteric activation by chorismate (Poulsen and Verpoorte, 1991). One chorismate mutase isozyme is inhibited by phenylalanine or tyrosine and activated by tryprophan, whereas the second is not affected by any of the aromatic amino acids. 

In one experiment washed chloroplasts were isolated and assayed for nitrite reductase, DAHP synthase-Mn and chorismate mutase-1 activities (Table 3). Since enzymes may fractionate with organelles by non-specific (or specific) association with the organelle surface, latency determinations were made. With this approach, activity determinations are made before and after rupture of the washed chloroplasts. If activities are located within the organelle, they are expected to increase dramatically following organelle disruption. Thus, nitrite reductase (chloroplast marker enzyme) gave a latency value of 16, a value similar to those obtained for DAHP synthase-Mn and chorismate mutase-1. The identity of chorismate mutase as the CM-1 isozyme was confirmed by its sensitivity to inhibition by L-tyrosine. 

Fig. 8. Ability of analogs of L-tyrosine (left chart) and of L-phenylalanine (right chart) to inhibit chorismate mutase-1 isolated from N. silvestris. ...

Fig. 9. Sequential pattern of allosteric control over biosynthesis of aromatic amino acids in the plastid compartment. In the presence of excess aromatic amino acids, L-tyrosine (TYR) inhibits arogenate dehydrogenase, L-phenylalanine (PHE) inhibits arogenate dehydratase and L-tryptophan (TRP) inhibits anthranilate synthase. The three aromatic amino acids exert allosteric inhibition (-) or activation (+) effects upon chorismate mutase-1 as symbolized. However, activation dominates over inhibition. The outcome of these events is to trap L-arogenate (AGN) between the various foci of control in the pathway. As shown symbolically, -arogenate (AGN) then acts to feedback inhibit DAHP synthase-Mn.

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