Rat liver .gamma.-butyrobetaine hydroxylase catalyzed reaction: influence of potassium, substrates, and substrate analogs on hydroxylation and decarboxylation

Abstract

Interaction of rat liver gamma-butyrobetaine hydroxylase (EC 1.14.11.1) with various ligands was studied by following the decarboxylation of alpha-ketoglutarate, formation of L-carnitine, or both. Potassium ion stimulates rat liver gamma-butyrobetaine hydroxylase catalyzed L-carnitine synthesis and alpha-ketoglutarate decarboxylation by 630% and 240%, respectively, and optimizes the coupling efficiency of these two activities. Affinities for alpha-ketoglutarate and gamma-butyrobetaine are increased in the presence of potassium. gamma-Butyrobetaine hydroxylase catalyzed decarboxylation of alpha-ketoglutarate was dependent on the presence of gamma-butyrobetaine, L-carnitine, or D-carnitine in the reaction and exhibited Km(app) values of 29, 52, and 470 microM, respectively. gamma-Butyrobetaine saturation of the enzyme indicated a substrate inhibition pattern in both the assays. Omission of potassium decreased the apparent maximum velocity of decarboxylation supported by all three compounds by a similar percent. beta-Bromo-alpha-ketoglutarate supported gamma-butyrobetaine hydroxylation, although less effectively than alpha-ketoglutarate. The rat liver enzyme was rapidly inactivated by 1 mM beta-bromo-alpha-ketoglutarate at pH 7.0. This inactivation reaction did not show a rate saturation with increasing concentrations of beta-bromo-alpha-ketoglutarate. None of the substrates or cofactors, including alpha-ketoglutarate, protected the enzyme against this inactivation. Unlike beta-bromo-alpha-ketoglutarate, beta-mercapto-alpha-ketoglutarate did not replace alpha-ketoglutarate as a cosubstrate. Both beta-mercapto-alpha-ketoglutarate and beta-glutathione-alpha-ketoglutarate were noncompetitive inhibitors with respect to alpha-ketoglutarate.

Synthesis of 2-oxoglutarate derivatives and their evaluation as cosubstrates and inhibitors of human aspartate/asparagine-β-hydroxylase

“…For the C3 benzyl substituted 2OG derivatives 21-25 with differently substituted phenyl rings, the substitution pattern on the phenyl rings appears to not affect the inhibitor potency within experimental error (Table 1, entries 6-10). With the exception of 4-methyl-2OG (26), which did not inhibit AspH (Table 1, entry 11), 2OG derivatives bearing substituents at the 2OG C4-position (Table 1, entries 12-18) were substantially more potent in inhibiting AspH than those bearing substituents at the C3-position: their relative potencies increased by a factor of 2 (for C3/4-Et) to $70 (for C3/4-CH 2 CH 2 C(CH 3 ) 3 ). The IC 50values of the C4-substituted 2OG derivatives range between 0.2 and 0.7 mM (Table 1, entries 12-18) and did not appear to depend on length or bulk of the tested C4-substituents.…”

“…Several Fe(II)-chelators have been previously identied to inhibit AspH in inhibitor screens. 41a The phenyl ring regioisomers 39, 40, and 41, which are 2OG derivatives bearing an aromatic core, did not inhibit AspH (Table 1, entries [24][25][26], as it was the case for the other tested phenyl ring containing 2OG derivatives 42-46 (Table 1, entries [27][28][29][30][31]. The 2OG derivative 48 whose carbon scaffold was not based on glutarate, but derived from succinate, inhibited AspH with moderate efficiency (IC 50 $ 3.3 mM; Table 1, entry 33).…”

“…To our knowledge, only a limited number of studies describing how 2OG derivatives bearing substituents at the C3- and/or C4-position modulate the activities of 2OG oxygenases ( i.e. human JmjC histone N ε -methyl lysine demethylase 4A, KDM4A; 25 human factor inhibiting the hypoxia-inducible transcription factor HIF-α, FIH; 24 and rat γ-butyrobetaine dioxygenase, BBOX 26 ) are reported. In part, this likely reflects a lack of a simple synthetic method to access these types of 2OG derivatives.…”

Effect of aromatic ring–containing drugs on carnitine biosynthesis in rats with special regard to p-aminomethylbenzoic acid

“…However, the question arises, how can this change in potassium cause 1 order of magnitude decrease in K m value of a-KG? Doubt is raised by the data provided by the authors, which show that the enzyme activity at 40 mmol/L K + reached the maximum at 100 mmol/L [22]. It should also be considered that a lesser part of total cellular a-KG is located in the cytosol, at the location of the Bu hydroxylase enzyme.…”

“…There is controversy about this value in the literature. The first article [20] reported 0.5 mmol/L K m for a-KG (using 40 mmol/L K + ion concentration), whereas a recent one [22] calculated 0.05 mmol/L (using 100 mmol/L K + ion). The latter authors attributed the difference to the K + ion concentration.…”