Publications

• Benzoyl-coenzyme A:glycine N-acyltransferase and phenylacetyl-coenzyme A:glycine N-acyltransferase from bovine liver mitochondria. Purification and characterization.

  Nandi D.L., Lucas S.V., Webster L.T. Jr.

  Two closely related acyl-CoA:amino acid N-acyl-transferases were purified to near-homogeneity from preparations of bovine liver mitochondria. Each enzyme consisted of a single polypeptide chain with a molecular weight near 33,000. One transferase was specific for benzoyl-CoA, salicyl-CoA, and cert ... >> More

  Two closely related acyl-CoA:amino acid N-acyl-transferases were purified to near-homogeneity from preparations of bovine liver mitochondria. Each enzyme consisted of a single polypeptide chain with a molecular weight near 33,000. One transferase was specific for benzoyl-CoA, salicyl-CoA, and certain short straight and branched chain fatty acyl-CoA esters as substrates while the other enzyme specifically used either phenylacetyl-CoA or indoleacetyl-CoA. Acyl-CoA substrates for one transferase inhibited the other. Glycine was the preferred acyl acceptor for both enzymes but either L-asparagine or L-glutamine also served. Peptide products for each transferase were identified by mass spectrometry. Enzymatic cleavage of acyl-CoA was stoichiometric with release of thiol and formation of peptide product. Apparent Km values were low for the preferred acyl-CoA substrates relative to the amino acid acceptors (10(-5) M range compared to greater than 10(-3) M). Both enzymes were inhibited by high nonphysiological concentrations of certain divalent cations (Mg2+, Ni2+, and Zn2+). In contrast to benzoyltransferase, phenylacetyltransferase was sensitive to inhibition by either 10(-4) M 5,5'-dithiobis(2-nitrobenzoate) or 10(-5) M p-chloromercuribenzoate; 10(-4) M phenylacetyl-CoA partially protected phenylacetyltransferase against 5,5'-dithiobis(2-nitrobenzoate) inactivation but 10(-1) M glycine did not. For activity, phenylacetyltransferase required addition of certain monovalent cations (K+, Rb+, Na+, Li+, Cs+, or (NH4)+) to the assay system but benzoyltransferase did not. Preliminary kinetic studies of both transferases were consistent with a sequential reaction mechanism in which the acyl-CoA substrate adds to the enzyme first, glycine adds before CoA leaves, and the peptide product dissociates last. << Less

  J. Biol. Chem. 254:7230-7237(1979) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Benzoyl-CoA: amino acid and phenylacetyl-CoA: amino acid N-acyltransferases.

  Webster L.T. Jr.

  Methods Enzymol 77:301-308(1981) [PubMed] [EuropePMC]

• Enzymatic characterization and elucidation of the catalytic mechanism of a recombinant bovine glycine N-acyltransferase.

  Badenhorst C.P., Jooste M., van Dijk A.A.

  Glycine conjugation, a phase II detoxification process, is catalyzed by glycine N-acyltransferase (GLYAT; E.C. 2.3.1.13). GLYAT detoxifies various xenobiotics, such as benzoic acid, and endogenous organic acids, such as isovaleric acid, which makes GLYAT important in the management of organic acid ... >> More

  Glycine conjugation, a phase II detoxification process, is catalyzed by glycine N-acyltransferase (GLYAT; E.C. 2.3.1.13). GLYAT detoxifies various xenobiotics, such as benzoic acid, and endogenous organic acids, such as isovaleric acid, which makes GLYAT important in the management of organic acidemias in humans. We cloned the open reading frame encoding the bovine ortholog of GLYAT from bovine liver mRNA into the bacterial expression vector pColdIII. The recombinant enzyme was expressed, partially purified, and enzymatically characterized. Protein modeling was used to predict Glu²²⁶ of bovine GLYAT to be catalytically important. This was assessed by constructing an E226Q mutant and comparing its enzyme kinetics to that of the wild-type recombinant bovine GLYAT. The Michaelis constants for benzoyl-CoA and glycine were determined and were similar for wild-type recombinant GLYAT, E226Q recombinant GLYAT, and GLYAT present in bovine liver. At pH 8.0, the E226Q mutant GLYAT had decreased activity, which could be compensated for by increasing the reaction pH. This suggested a catalytic mechanism in which Glu²²⁶ functions to deprotonate glycine, facilitating nucleophilic attack on the acyl-CoA. The recombinant bovine GLYAT enzyme, combined with this new understanding of its active site and reaction mechanism, could be a powerful tool to investigate the functional significance of GLYAT sequence variations. Eventually, this should facilitate investigations into the impact of known and novel sequence variations in the human GLYAT gene. << Less

  Drug Metab. Dispos. 40:346-352(2012) [PubMed] [EuropePMC]

• Purification to homogeneity of mitochondrial acyl CoA:glycine N-acyltransferase from human liver.

  Mawal Y.R., Qureshi I.A.

  Mitochondrial acyl CoA:glycine N-acyl transferase (ACGNAT) was purified to homogeneity from adult human liver. It was found to be a monomer of 30 kD, having a pI of 6.8. ACGNAT retained 47% of enzymatic activity at 100 mM NaCl concentration, whereas 21% of the activity was retained with KCl and 32 ... >> More

  Mitochondrial acyl CoA:glycine N-acyl transferase (ACGNAT) was purified to homogeneity from adult human liver. It was found to be a monomer of 30 kD, having a pI of 6.8. ACGNAT retained 47% of enzymatic activity at 100 mM NaCl concentration, whereas 21% of the activity was retained with KCl and 32% with K3PO4 at 100 mM concentration as compared to the control. The stability studies revealed no change in activity at 4 degrees C for up to 72 h, 25 degrees C for 4 h and at 37 degrees C for 1 h. The Km values of human ACGNAT for benzoyl CoA, salicyl CoA, isovaleryl CoA and octanoyl CoA were 57.9, 83.7, 124 and 198 mM, respectively, and the corresponding Vmax values were 17.1, 10.1, 7.64 and 3.3 mumol/min/mg protein. The availability of pure human ACGNAT would help in studying the molecular genetics and structural biology of this protein which is important in the detoxification of various endogenous and xenobiotic acyl CoA's. << Less

  Biochem. Biophys. Res. Commun. 205:1373-1379(1994) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The utilization of alanine, glutamic acid, and serine as amino acid substrates for glycine N-acyltransferase.

  van der Westhuizen F.H., Pretorius P.J., Erasmus E.

  The conjugation of benzoyl-CoA with the aliphatic and acidic amino acids by glycine N-acyltransferase, as well as the amides of the latter group, was investigated. Bovine and human liver benzoyl-amino acid conjugation were investigated using electrospray ionization tandem mass spectrometry (ESI-MS ... >> More

  The conjugation of benzoyl-CoA with the aliphatic and acidic amino acids by glycine N-acyltransferase, as well as the amides of the latter group, was investigated. Bovine and human liver benzoyl-amino acid conjugation were investigated using electrospray ionization tandem mass spectrometry (ESI-MS-MS). Bovine glycine N-acyltransferase catalyzed conjugation of benzoyl-CoA with Gly (Km(Gly) = 6.2 mM), Asn (Km(Asn) = 129 mM), Gln (Km(Gln) = 353 mM), Ala (Km(Ala) = 1573 mM), Glu (Km(Glu) = 1148 mM) as well as Ser in a sequential mechanism. In the case of the human form, conjugation with Gly (Km(Gly) = 6.4 mM), Ala (Km(Ala) = 997 mM), and Glu was detected. The presence of these alternative conjugates did not inhibit bovine glycine N-acyltransferase activity significantly. Considering the relatively low levels at which these conjugates are formed, it is unlikely that they will have a significant contribution to acyl-amino acid conjugation under normal conditions in vivo. However, their cumulative contribution to acyl-amino acid conjugation under metabolic disease states may prove to have a useful contribution to detoxification of elevated acyl-CoAs. << Less

  J. Biochem. Mol. Toxicol. 14:102-109(2000) [PubMed] [EuropePMC]