Chorismate mutase strain

Scheme 4.12 Catalytic antibody 1F7 was raised against the transition state analog 28 and possesses modest chorismate mutase activity. It can complement a permissive yeast strain that is auxotrophic for phenylalanine and tyrosine by replacingthe natural enzyme (CM) in the shikimate biosynthetic pathway.

Ranaghan KE, L Ridder, B Szefczyk, WA Sokalski, JC Hermann, AJ Mulholland (2004) Transition state stabilization and substrate strain in enzyme catalysis ab initio QM/MM modelling of the chorismate mutase reaction. Organic Biomolecular Chemistry 2 (7) 968-980... 

Over the past decade, several strains of yeast [43, 44] and E. coli [45, 46] have been engineered that lack chorismate mutase. A typical bacterial selection system is depicted schematically in Fig. 3.5. It is based on E. coli strain KA12 [45], which has deletions of the chromosomal genes for both bifunctional chorismate mutases (chorismate mutase-prephenate dehydrogenase and chorismate mutase-prephenate dehydratase). Monofunctional versions of prephenate dehydratase [47] and prephenate dehydrogenase [48] from other organisms are supplied by the plasmid pKIMP-UAUC, leaving the cells deficient only in chorismate mutase activity [45]. 

The Fab fragment of 1F7 has already been shown to function in the cytoplasm of a chorismate mutase-deficient yeast strain [43,44]. When produced at a sufficiently high level, the catalytic antibody is able to replace the missing enzyme and weakly complement the metabolic defect. Conceivably, therefore, it can be placed under selection pressure to identify variants that have higher catalytic efficiency. Preliminary results from such experiments appear quite promising [69]. 

The calculated barrier to reaction in chorismate mutase was 17.8 kcal/mol, compared to 42 kcal/mol in the gas phase. Factors other than substrate distortion also play an important part in reducing the barrier to reaction in the enzyme important interactions were identified by a simple decomposition analysis (as described in sections 6.1 and 6.2 above). It was found that Glu78 and Arg90 specifically stabilize the transition state, relative to the bound substrate [8]. Overall, therefore, catalysis in chorismate mutase can be rationalized in terms of a combination of substrate strain and transition state stabilization. While it is possible to analyse all these catalytic effects as arising from maximal binding in the enzyme being achieved at the transition state, it appears useful to separate the different types of contribution. The possible role of substrate destabilization/distortion or strain in lowering the barrier to reaction in enzyme reactions, as put forward by Haldane [219], and invoked in... 

The exact mechanism of the action by chorismate mutase is stUl not clear, in spite of extensive experimental and theoretical investigations. Several suggestions have been proposed concerning the origin of the catalysis. They include (a) the stabilization of transition state by the enzyme, presumably through electrostatic interactions from the active site residues (b) the promotion of substrate conformational transition to generate the reactive CHAIR conformer at the active site (see Scheme 1.2) (c) the increase of populations of near attack conformers (NACs) and (d) strain effects and conformational compression. These proposals will be discussed below. 

Strains and conformational compression of the substrate at the enzyme active site were also proposed to explain the rate acceleration by chorismate mutase [66, 70,... 

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