Purification, characterization and inhibition of d‐3‐aminoisobutyrate aminotransferase from the rat liver

Abstract

D-3-Aminoisobutyrate -pyruvate aminotransferase was purified 2000-fold from rat liver extract using heat treatment, ammonium sulfate fractionation, carboxylmethyl-Sepharose CL-6B, DEAE-Sepharose CL-6B, hydroxyapatite, Sephacryl S-200 and electrofocusing chromatographies. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 220 kDa and the subunit molecular mass was 52 kDa. The enzyme exhibited absorption maxima at 280 nm and 412 nm with a shoulder at 330 nm at neutral pH. The pH optimum for enzyme activity was 9.5 and the K, values for p-alanine and pyruvic acid were calculated to be 0.81 mM and 0.45 mM, respectively. The purified enzyme catalyzed the transamination of w-amino acids; P-alanine and D-3-aminoisobutyric acid served as good amino donors, and pyruvic acid, glyoxylic acid and oxaloacetic acid were favorable amino acceptors.6-Azauracil and 6-azathymine were found to be potent inhibitors of purified rat liver D-3-aminoisobutyratepyruvate aminotransferase. 6-Azauracil acted as a competitive inhibitor with respect to p-alanine, and was an uncompetitive inhibitor with respect to pyruvic acid with a Ki of approximately 8.9 mM.

AGXT2: a promiscuous aminotransferase

“…Several small molecule inhibitors of AGXT2 have already been described [3, 4, 7, 10, 69, 70], some of which are endogenous molecules and others are analogs of drugs or toxins. Further studies are needed to better characterize the known inhibitors and develop new lead compounds that selectively inhibit a subset of the specific metabolic pathways regulated by AGXT2 without inhibiting its other enzymatic activities.…”

“…Because AGXT2 catalyzes multiple aminotransferase reactions, several alternative names for this enzyme have been proposed. AGXT2 has been referred to as D-3-aminoisobutyrate-pyruvate aminotransferase [3], (R)-3-amino-2-methylpropionate-pyruvate aminotransferase [4] and BAIB-pyruvate aminotransferase [5] due to its ability to utilize D-β-aminoisobutyric acid (BAIB) as an amino group donor (Figure 1B). It has been called dimethylarginine-pyruvate aminotransferase due to its ability to utilize methylarginines such as asymmetric dimethylarginine (ADMA) as amino group donors [6] (Figure 1C), and alanine- γ , δ -dioxovaleric acid aminotransferase because it can catalyze the transfer of an amino group from alanine to γ,δ-dioxovaleric acid (DOVA) [7] (Figure 1D).…”

Severe child form of primary hyperoxaluria type 2 - a case report revealing consequence of GRHPR deficiency on metabolism

“…The production of glyoxylate in mitochondria by the HOGA enzyme and its detoxification in this compartment is less satisfactorily explained. Based on known data, it can be inferred that except for the GRHPR enzyme, mitochondrial AGXT2 can effectively use pyruvate and glyoxylate also for the transamination of D-3-aminoisobutyrate (D-BAIB) and β-alanine (BAL) [26]. It has been long established that genetic factors influence the high excretion of BAIB, originated preferentially from the degradation of thymine (R- form) and to a lesser extent from valine (S-form) [27–30].…”

“…This enzyme catalyses the transamination of non-stereospecific BAL, GABA and from stereospecific isomers only L-BAIB with 2-oxoglutarate [33, 34]. In a report subsequently published by Tamaki [26], a mixture of appropriate substrates and enzymes AGXT2 and ABAT provide in vitro condition conversion between S-BAIB and R-BAIB. In context with genetic abnormality (AGXT2 deficiency), we consider changes of the mitochondrial pool of HOGA, glyoxylate, and their additive inhibitory effects on GRHPR that accentuate deficiency.…”

Distribution of D-3-aminoisobutyrate-pyruvate aminotransferase in the rat brain

“…We, as well as others, have previously purified and characterized D-AIB AT in the rat liver [1,2], and its gene structure was determined by Matsui-Lee et al [3]. The condition resulting in high amounts of D-AIB excretion in the urine is inherited in an autosomal recessive fashion [4], and individuals with this condition have been reported to lack D-AIB AT activity [5].…”