Amine acetylating enzyme

Amine Acetylation. The first acetylation reaction to be studied intensively was the acetylation of aromatic amines, of which sulfanilamide was used as a representative (II). This system was used for the first assays of CoA. The amine-acetylating enzyme has been partially purified. 

The loss of acetyl CoA was found to be caused by a nonenzymatic transfer of acetyl groups to other sulfhydryl compounds added to the system. Ordinarily sulfhydryl compounds should not be required in reactions of acetyl CoA, as opposed to systems using free CoA, whose —SH group needs protection. But the amine-acetylating enzyme also has essential —SH groups that require protection. The dilemma was... 

Choline acetylase and the amine-acetylating enzyme are distinct, and do not cross react with the substrates of the other enzyme. 

Acetylation may occur with -NHg, -OH, and -SH groups. Acetylation of primary aromatic amines and sulfonamide nitrogens are the most important of these reactions in the inactivation of drugs. These reactions occur in most laboratory and domestic animals. The dog is unable to acetylate aromatic amines or hydrazines, probably due to a lack of selective acetylating enzymes. Deacetylation of the conjugate may occur in certain species, e.g. the chicken de-acetylates aromatic amines, and dogs deacetylate aliphatic amino acids. Acetylation reactions have also been reported to occur in amphibia, fish, and a few insect species [20]. 

CoA involved many simultaneous approaches. Many years were spent in unraveling the tangle of information obtained from studies on microbial nutrition, enzyme reactions (in addition to amine acetylation) that require CoA, yeast oxidation of acetate, and degradation and synthesis of the cofactor. The results of these studies are summarized in the following sections. 

The concept that enzymic acetylation is a stepwise process involving the formation and breakdown of an acetylated enzyme intermediate has been suggested by several investigators. Bessman and Lipmann (1953) observed that a pigeon liver preparation reversibly catalyzed the acetyl exchange between 4-acetaminoazobenzene-4 - sulfonic acid and various aryl amines including aniline, p-aminobenzoic acid, and sulfanilamide. They deduced that an acetyl-enzyme compound was formed as an intermediate... 

Figure 20.6 Available amine groups on an antibody molecule may be modified with the NHS ester end of SATA to produce amide bond derivatives containing terminal protected sulfhydryls. The acetylated thiols may be deprotected by treatment with hydroxylamine at alkaline pH. Reaction of the thiolated antibody with a maleimide-activated enzyme results in thioether crosslinks.

The enzyme can also catalyze the transfer of an acetyl group from an N-acetylated hydroxylamine (hydroxamic acid) to form an acetoxy product, i.e., an N to O transacetylation and this pathway does not require acetyl Co-A (12). A-hydroxy-4-acetylaminobiphenyl provides an example of this conversion as shown in Figure 7.7. The significance of this pathway is that it leads to the activation of the hydroxamic acid because acetoxy derivatives of aromatic amines are chemically reactive and many are carcinogens such as the heterocyclic amines formed when meat is heated to a high temperature, e.g., 2-amino-1-mcthyl-6-phenylirnidaz()[4,5-i ]pyri(linc. 

GG is used extensively for analysis of antidepressants (Orsulak et al, 1989), but HPLC assays and enzyme immunoassays have become more popular in recent years. However, GC has advantages such as economy and ready availability. LCD and NPD generally are the detectors of choice (Coutts and Baker, 1982). NPD is relatively efficient for the analysis of tricyclic antidepressants (TCAs) as derivatization is not necessary, although the secondary, demethylated amines are sometimes derivatized to improve resolution and peak shape (Coutts and Baker, 1982). Acetylation, under aqueous or anhydrous conditions, followed by GC-NPD, has been used extensively for analysis of TCAs and the tetracyclic antidepressant maprotiline in plasma samples (Drebit et al., 1988). O Table 1-1 summarizes GC assays for some commonly prescribed antidepressants and their metabolites. 

This enzyme [EC 2.3.1.5], also known as acetyl-CoA arylamine A-acetyltransferase and arylamine acetylase, catalyzes the reaction of acetyl-CoA with an arylamine to produce coenzyme A and an A-acetylarylamine. This enzyme exhibits a low specificity with respect to the aromatic amine substrate. In fact, even serotonin can serve as a substrate. The enzyme has also been reported to catalyze acetyl-transfer reactions between arylamines without the use of coenzyme A. 

This enzyme [EC 2.1.1.4], also referred to as acetylsero-tonin methyltransferase, catalyzes the reaction of 5-ad-enosyhnethionine with A-acetylserotonin to produce 5-adenosylhomocysteine and A-acetyl-5-methoxytrypt-amine. 

This enzyme [EC 5.4.2.S], also known as acetylglucos-amine phosphomutase and A -acetylglucosamine-phos-phate mutase, catalyzes the interconversion of M-acetyl-D-glucosamine 1-phosphate and A -acetyl-o-glucosamine 6-phosphate. The enzyme is activated by A -acetyl-o-glu-cosamine 1,6-bisphosphate. 

Polyamine oxidase (amine oxygen oxidoreductase, deaminating, flavin-containing), is also a FAD-dependent enzyme and has many similarities to MAO. It is responsible for the oxidation of the secondary amino group in such substrates as A/-acetyl spermine and spermidine in the biosynthesis of spermidine and putrescine [1,12], This enzyme will not be covered in this chapter. 

The S-CoA derivative then acylates the amino group of the particular amino acid in an analogous way to the acetylation of amine groups described above, yielding a peptide conjugate. This is catalyzed by an amino acid N-acyltransferase, which is located in the mitochondria. Two such enzymes have been purified, each using a different group of CoA derivatives. 

---