An investigation into the origin of acyl-amino acid conjugation in the human and bovine metabolism

Abstract

Glycine conjugation and subsequent excretion of carboxylic acids of endogenous and exogenous origin is a normal detoxication process. A wide variety of short- and medium-chain, mono and dicarboxyl, acylglycine conjugates are also excreted in the urine of patients suffering from various metabolic disorders. The formation of these conjugates is catalyzed by glycine N-acyltransferase (EC 2.3.1.13). During the last two decades acylarnino acid conjugates with amino acid moieties other than glycine have also been detected in the urine of patients with metabolic disorders. These include propionylalanine, propionylglutamic acid, benzoylalanine, isovalerylalanine, isovalerylglutamic acid, and 3-methylcrotonylglutamic acid. This study investigated the conjugation of benzoyl-CoA with the aliphatic and acidic amino acids as well as their amides by glycine N-acyltransferase. Bovine and human glycine N-acyltransferase was purified from liver mitochondria in a five-step strategy and conjugation was investigated using these preparations in addition to mitochondrial lysates. A new electrospray ionization tandem mass spectrometry-based method was developed to characterize as well as quantify benzoylamino acid conjugation. Bovine glycine N-acyltransferase conjugated benzoyl-CoA with glycine (KmGiy = 6.2 mM), asparagine (KmAsn = 129 mM), glutamine (KmGJn = 353 mM), alanine (KmAJa = 1573 mM), and glutamic acid (KmGiu = 1148 mM) in a sequential mechanism. Benzoyl conjugation with serine was also detected. Human glycine N-acyltransferase, on the other hand, showed a less diverse amino acid substrate utilization and conjugated benzoyl-CoA with glycine (KmGiy = 6.4 mM), alanine (KmAJa = 998 mM), as well as glutamic acid. The results indicated that, compared to glycine, all other amino acids conjugated with benzoyl-CoA at much lower rates, which explains the relative low levels of the newly detected conjugates in urine. Inhibition of human and bovine glycine N-acyltransferase by benzoylamino acid conjugates other than glycine were much lower compared to benzoylglycine and it is unlikely to have an inhibitory effect on glycine N-acyltransferase in vivo.

A human liver cDNA clone was obtained using bovine N-terminal sequence data which shared a strong homology (82 % ) with bovine GNAT cDNA as well as bovine glutarnine N-phenylacetyltransferase cDNA (77 %). Attempts to express the cDNA in E.coli were not successful and the identity of this cDNA could therefore not be confirmed.