Serine nitrogen removal

Serine itself would be insufficiently nucleophilic to attack the ester carbonyl, so the reaction is facilitated by participation of the imidazole ring of histidine. The basic nitrogen in this residue is oriented so that it can remove a proton from the serine hydroxyl, increasing nucleophilicity and allowing attack on the ester carbonyl. This leads to formation of the transient acetylated enzyme, and release of choline. Hydrolysis of the acetylated enzyme utilizes water as nucleophile, but again involves the imidazole ring, and regenerates the free enzyme. 

Not included in this simplified description of the active site is the important role played by another amino acid residue. The basicity of the histidine nitrogen is increased because of the proximity of a neighbouring aspartate residue. This facilitates removal of a proton from the active site serine. The relationship of these three residues... 

Although the nitrogen atoms of most amino acids are transferred to a-ketoglutarate before removal, the a-amino groups of serine and threonine can be directly converted into NH4 +. These direct deaminations are catalyzed by serine dehydratase and threonine dehydratase, in which PLP is the prosthetic group. 

The combined ether extract (which may be washed with 0.01 N HCl to remove peptide material) is evaporated to dryness with a stream of nitrogen and 50 /il of 1 N HCl is added. After mixing, the thiazolinone derivative of the NH2 terminal residue is converted to the phenyl-thiohydantoin by incubation at 80°C (temperature block) for 10 min. If Asx or Glx residues are expected, 80°C for 3 min may give better results and for threonine, proline and serine, 50°C for 10 min may be better (Li and Yanofsky 1972). After cooling, the phenylthiohydantoin is extracted into ethyl acetate (100-200 /rl for each of 3 extractions), which is then evaporated with a stream of nitrogen. The phenylthio-hydantoins of arginine, histidine and cysteic acid will usually remain in the aqueous phase and may be recovered by lyophilization and dissolving the residue in methanol. 

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