.alpha.-Ketoglutaric acid: solution structure and the active form for reductive amination by bovine liver glutamate dehydrogenase

Abstract

A study of the various solution forms of alpha-ketoglutaric acid using UV absorption spectrophotometry and 13C NMR spectroscopy shows that at neutral pH alpha-ketoglutarate exists predominantly as the keto form with about 7% hydrated form (gem-diol) and a small amount of cyclic form. Protonation of the gamma-carboxylate group increases the amount of cyclic form to 20% with very little increase in the amount of gem-diol. Protonation of the alpha-carboxylate increases the amount of cyclic form to 30% and the amount of gem-diol to 35%. The pH and temperature dependence of the 13C NMR line widths indicated that the interconversion of keto and cyclic forms is extremely rapid. The rate of interconversion of keto and gem-diol forms was studied spectrophotometrically at various temperatures, buffers, and pH values and by varying the total concentration of alpha-ketoglutarate. The hydration reaction of alpha-ketoglutarate is not catalyzed by bovine liver glutamate dehydrogenase (E) or by the E-NADPH complex. The enzyme uses the keto form of alpha-ketoglutarate as the substrate. The gem-diol form is not itself a substrate but becomes converted to the keto form with a half-life of about 0.3 min at 15 degrees C in 0.1 M potassium phosphate buffer (pH 7.6).

Lactate is a mediator of metabolic cooperation between stromal carcinoma associated fibroblasts and glycolytic tumor cells in the tumor microenvironment

“…4C. In both, hMSCs and CAFs, a signal at 36.9 ppm, assigned to 13 C-labeled alpha-ketoglutarate (C-3 position), based on the chemical shift [28] and the fate of the 13 C-3 label of lactate [29], is visible for cells incubated with sodium- l -lactate-3- 13 C. Additional signals visible in the full 13 C NMR spectra were from the concentration and reference standard DSS, and ${\mathrm{HCO}}_3^{-}$. hMSCs were able to survive and grow in media depleted of glucose but supplemented with 5 or 15 mM lactate (Fig.…”

“…Two samples each of hMSCs and CAFs were incubated with 10 mM sodium- l -lactate- 13 C-3 for 4 h followed by PCA extraction. Proton-decoupled 13 C NMR spectroscopy revealed 13 C labeling of a metabolite, assigned to α-ketoglutarate- 13 C-3(α-KG), based on its 13 C chemical shift [28], for all 4 samples (2 each for hMSCs and CAFs) incubated with 13 C-labeled lactate but not in the untreated hMSC sample (control). An additional signal, detected in all samples outside of the spectral region shown here, was assigned to ${\mathrm{HCO}}_3^{-}$.…”

Kinetics of Cytochrome P450 2E1-Catalyzed Oxidation of Ethanol to Acetic Acid via Acetaldehyde

“…The form of a carbonyl used as substrate is a general problem with all enzymes utilizing aldehydes and ketones and is not trivial to answer except under special conditions (50,51). An explanation for the scheme is that the I and Q forms (Figs.…”

Carbonyl oxygen exchange evidence of imine formation in the glutamate dehydrogenase reaction and identification of the "occult role" of NADPH.

“…However, we have previously reported (11) the rate of the diol-toketone reaction of a-ketoglutarate, and that rate is too slow to account for the observed rate of exchange by 2 orders of magnitude. It is also useful to note in this connection that adihydroxyglutarate is not a substrate for this enzyme (11).…”