The Effect of 2-Ketobutyrate on Mitochondrial Substrate-Level Phosphorylation

Abstract

The reaction catalyzed by succinate-CoA ligase in the mitochondrial matrix yields a high-energy phosphate when operating towards hydrolysis of the thioester bond of succinyl-CoA, known as mitochondrial substrate-level phosphorylation (mSLP). The catabolism of several metabolites converge to succinyl-CoA but through different biochemical pathways. Among them, threonine, serine and methionine catabolize to succinyl-CoA through the common intermediate, 2-ketobutyrate. During the course of this pathway 2-ketobutyrate will become succinyl-CoA through propionyl-CoA catabolism, obligatorily passing through an ATP-consuming step substantiated by propionyl-CoA carboxylase. Here, by recording the directionality of the adenine nucleotide translocase while measuring membrane potential we tested the hypothesis that catabolism of 2-ketobutyrate negates mSLP due to the ATP-consuming propionyl-CoA carboxylase step in rotenone-treated, isolated mouse liver and brain mitochondria. 2-Ketobutyrate produced a less negative membrane potential compared to NADH or FADH2-linked substrates, which was sensitive to inhibition by rotenone, atpenin and arsenate, implying the involvement of complex I, complex II and a dehydrogenase—most likely branched chain keto-acid dehydrogenase, respectively. Co-addition of 2-ketobutyrate with NADH- or FADH2-linked substrates yielded no greater membrane potential than in the presence of substrates alone. However, in the presence of NADH-linked substrates, 2-ketobutyrate prevented mSLP in a dose-dependent manner. Our results imply that despite that 2-ketobutyrate leads to succinyl-CoA formation, obligatory metabolism through propionyl-CoA carboxylase associated with ATP expenditure abolishes mSLP. The provision of metabolites converging to 2-ketobutyrate may be a useful way for manipulating mSLP without using pharmacological or genetic tools.

Circulating markers of NADH-reductive stress correlate with mitochondrial disease severity

“…We also noted metabolites in the anaplerotic pathway leading from propionyl-CoA to the TCA cycle (Supplemental Figure 2A) showed discordant changes: patients with MELAS showed a significant accumulation of C3-carnitine but a decrease in succinate, just missing our FDR threshold (adjusted P = 0.05), while the intermediate, methylmalonate (MMA), was not significantly different. We hypothesize that this pattern is consistent with inhibition of the propionyl-CoA carboxylase reaction (Supplemental Figure 2A) and is supported by prior studies of this pathway, though the specific mechanism remains unclear (69).…”

“Fuel for the Damage Induced”: Untargeted Metabolomics in Elite Rugby Union Match Play

“…The reduced salivary purine profiles due to oxidative stress have been observed post exhaustive exercise in male athletes [ 39 ]. The increase in serum 2-hydroxybutyrate post-match is indicative of the cumulative stress upon energy systems [ 40 ] and may be due to increased catabolism of l -threonine [ 41 ] and glutathione synthesis [ 42 ] in response to oxidative stress. Transient increases have also been witnessed after HIT exercise [ 9 ] and proposed as a marker of dysglycemia [ 43 ].…”

Serum Metabolomic Profiling to Reveal Potential Biomarkers for the Diagnosis of Fatty Liver Hemorrhagic Syndrome in Laying Hens

“…As the substrate of protein synthesis, gluconeogenesis, and ketone synthesis, amino acids can also provide energy through the TCA cycle ( Lopansri et al, 2006 ; Miao et al, 2014 ). Isoleucine, serine, and threonine are the precursors of succinyl coenzyme A, the intermediate metabolite of TCA cycle ( Bui et al, 2019 ). In this experiment, the increase of serine content and the decrease of isoleucine and threonine content indicate the disorder of amino acid metabolism.…”