Chorismate mutase values

QM/MM methods have proved their value for enzyme reactions in differentiating between alternative proposed mechanisms, and in analysing contributions to catalysis. A current example is the analysis of the contribution of conformational effects and transition state stabilization in the reaction catalysed by the enzyme chorismate mutase.98,99 QM/MM calculations can be performed with... 

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-... 

Chorismate mutase provides an example of an enzyme where QM/MM calculations have identified an important catalytic principle at work [8], This enzyme catalyses the Claisen rearrangement of chorismate to prephenate. The reaction within the enzyme is not believed to involve chemical catalysis, and this pericylic reaction also occurs readily in solution. Lyne et al. [8] investigated the reaction in chorismate mutase in QM/MM calculations, at the AMI QM level (AMI was found to perform acceptably well for this reaction in comparisons with ab initio results for the reaction in the gas phase [8]). Different sizes of QM system were tested in the QM/MM studies (e.g. including the substrate and no, or up to three, protein side chains), and similar results found in all cases. The reaction was modelled by minimization along an approximate reaction coordinate, defined as the ratio of the forming C-C and breaking C-0 bonds. Values of the reaction coordinate were taken from the AMI intrinsic reaction coordinate for the gas-phase reaction. 

Chorismic acid is the key branch point intermediate in the biosynthesis of aromatic amino acids in microorganisms and plants (Scheme 1.1a) [1]. In the branch that leads to the production of tyrosine and phenylalanine, chorismate mutase (CM, chorismate-pyruvate mutase, EC 5.4.99.5) is a key enzyme that catalyzes the isomerization of chorismate to prephenate (Scheme 1.1b) with a rate enhancement of about lO -lO -fold. This reaction is one of few pericyclic processes in biology and provides a rare opportunity for understanding how Nature promotes such unusual transformations. The biological importance of the conversion from chorismate to prephenate and the synthetic value of the Claisen rearrangement have led to extensive experimental investigations [2-43]. 

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. 

This realization has not escaped the bioorganic community, and a number of research groups, notably those of Andrews and Berchtold, have synthesized molecules that are designed to mimic the presumed bicyclic transition state. A truly potent inhibitor has eluded our grasp, however, as reflected by the fact that most compounds investigated bind more weakly to chorismate mutase/prephenate dehydrogenase than does chorismate (as reflected by the substrate Km value). 1-Adamantyl phosphonic acid is the most potent inhibitor previously reported, with an I50 value twenty-fold less than chorismate Km under comparable conditions. 

In view of the non-linear kinetics frequently observed with chorismate mutases and the resultant difficulty of determining true values, most inhibitor binding constants are reported as I50 values, equal to the concentration of inhibitor which produces 50% inhibition when the chorismate concentration equals Km. In Table 1, the compounds are compared as the ratio of reported Iso/Km values, since they were evaluated against enzymes from different sources and under different conditions. 

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