Enzymatic inversion

Applying physiological conditions regarding substrate concentration, solvent and temperature, the enzymatic inversion of sucrose is practically irreversible. 

Wechtei, W.J., Loughhead, D.G., Reishei, R.J., van Geissen, G.J. and Kaiser, D.G. (1974) Enzymatic inversion at saturated carbon nature and mechanism of the inversion of R(—)p-iso-butyl hydratropic acid. Biochem. Biophys. Res. Commun. 61 833-837. 

Table E.5.2. Inverse substrate concentration and inverse enzymatic rate calculation with and...

An interesting combination of enzymatic with non-enzymatic transformation in a one-pot three-step multiple sequence was reported by Waldmann and coworkers [82]. Phenols 125 in the presence of oxygen and enzyme tyrosinase are hydroxylated to catechols 126 which are then oxidized in situ to ortho quinones 127. These intermediates subsequently undergo a Diels-Alder reaction with inverse electron demand by reaction with different dienophiles (Table 4.19) to give endo bicyclic 1,2-diketones 128 and 129 in good yields. 

Stereoinversion Stereoinversion can be achieved either using a chemoenzymatic approach or a purely biocatalytic method. As an example of the former case, deracemization of secondary alcohols via enzymatic hydrolysis of their acetates may be mentioned. Thus, after the first step, kinetic resolution of a racemate, the enantiomeric alcohol resulting from hydrolysis of the fast reacting enantiomer of the substrate is chemically transformed into an activated ester, for example, by mesylation. The mixture of both esters is then subjected to basic hydrolysis. Each hydrolysis proceeds with different stereochemistry - the acetate is hydrolyzed with retention of configuration due to the attack of the hydroxy anion on the carbonyl carbon, and the mesylate - with inversion as a result of the attack of the hydroxy anion on the stereogenic carbon atom. As a result, a single enantiomer of the secondary alcohol is obtained (Scheme 5.12) [8, 50a]. 

The result of enzymatic decarboxylation was extremely clear. While (S)-compound resulted in C-containing product, (/ )-compound gave the product with C no more than natural abundance. Apparently, the enzyme decarboxylated pro-(/ ) carboxyl group selectively and the reaction proceeds with net inversion of configuration. Thus, the presence of a planar intermediate can be reasonably postulated. Enantioface-differentiating protonation to the intermediate will give the optically active final product (Eig. 12). 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

Miller and Wolfenden, 2002). This latter ratio is the inverse of the rate enhancement achieved by the enzyme. In other words, the enzyme active site will have greater affinity for the transition state structure than for the ground state substrate structure, by an amount equivalent to the fold rate enhancement of the enzyme (rearranging, we can calculate KJX = Ksik Jk, )). Table 2.2 provides some examples of enzymatic rate enhancements and the calculated values of the dissociation constant for the /A binary complex (Wolfenden, 1999). 

As already mentioned, macular zeaxanthin comprises two stereoisomers, the normal dietary (3/(,37()-/caxanthin and (3f ,3 S)-zeaxanthin(=(meyo)-zeaxanthin), of which the latter is not normally a dietary component (Bone et al. 1993) and is not found in any other compartment of the body except in the retina. The concentration of (tneso)-zeaxanthin in the retina decreases from a maximum within the central fovea to a minimum in the peripheral retina, similar to the situation with (3/ ,37 )-zeaxanthin. This distribution inversely reflects the relative concentration of lutein in the retina and gave rise to a hypothesis (Bone et al. 1997) that (meso)-zeaxanthin is formed in the retina from lutein. This was confirmed by an experiment in which xanthophyll-depleted monkeys had been supplemented with chemically pure lutein or (3/ ,37 )-zeaxanthin (Johnson et al. 2005). (Meyo)-Zeaxanthin was exclusively detected in the retina of lutein-fed monkeys but not in retinas of zeaxanthin-fed animals, demonstrating that it is a retina-specific metabolite of lutein only. The mechanism of its formation has not been established but may involve oxidation-reduction reactions that are mediated photochemically, enzymatically, or both. Thus, (meso)-zeaxanthin is a metabolite unique to the primate macula. 

An S Ar (nucleophilic substitution at aromatic carbon atom) mechanism has been proposed for these reactions. Both nonenzymatic and enzymatic reactions that proceed via this mechanism typically exhibit inverse solvent kinetic isotope effects. This observation is in agreement with the example above since the thiolate form of glutathione plays the role of the nucleophile role in dehalogenation reactions. Thus values of solvent kinetic isotope effects obtained for the C13S mutant, which catalyzes only the initial steps of these reactions, do not agree with this mechanism. Rather, the observed normal solvent isotope effect supports a mechanism in which step(s) that have either no solvent kinetic isotope effect at all, or an inverse effect, and which occur after the elimination step, are kinetically significant and diminish the observed solvent kinetic isotope effect. 

---