Substrate formation, organic residue

A third early mechanism for enzymic processes involves the formation of imines between the amino group of a lysine residue on an enzyme and the carbonyl group of a substrate, followed by standard imine chemistry. The first example concerned the decarboxylation of acetoacetic acid (Hamilton and Westheimer, 1959). The mechanism was based on the non-enzymic physical organic chemistry of Kai Pedersen (Pedersen, 1934). He postulated that the catalysis by aniline of the decarboxylation of dimethylacetoacetic acid proceeds by a mechanism parallel to that shown in Scheme 7 for acetoacetic acid itself (Pedersen, 1938). 

The standard reaction mixture contained 0.1 mL of 10 mM substrate dissolved in acetonitrile, 200 fiM NADPH, 0.1 mL of cytosol, and 10 mg/mL bovine serum albumin in 0.1 M potassium phosphate buffer (pH 6.2). The final volume was 2.0 mL. The reaction was continued at 25°C for 10 minutes before being terminated by the addition of 0.2 mL of 2 M HC1. The product was extracted twice with 5 mL of ethyl acetate in the presence of 50 nmol of 3-acetyl-7-dimethylaminocoumarin as the internal standard. After centrifugation, the organic layer was evaporated in vacuo. The residue was taken up in 1.0 mL of the mobile phase and a 5 /xL aliquot was used for HPLC analysis. The formation of both enantiomers was linear with time for up to 30 minutes. The rate of formation of the S enantiomer was about ninefold higher than that of the R enantiomer. 

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