PubMed · 8278806
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D E Cane. 1994-01-21. Polyketide biosynthesis: molecular recognition or genetic programming?. https://doi.org/10.1126/science.8278806
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Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of 13C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.
Although mammalian hibernators rely on stored body fat as a source of energy, direct measurement of energy substrate preference in heart tissue during hibernation, as well as potential mechanisms controlling fatty acid oxidation has not been examined. In order to determine whether an increase in fatty acid utilization occurs during hibernation, glucose and palmitate oxidation were measured in isolated working hearts from hibernating and non-hibernating Richardson's ground Squirrels. Hearts were perfused at either 37 degrees or 5 degrees C with perfusate containing 11 mM [U-14C]glucose and 1.2 mM [9,10-3H]palmitate, which allowed for direct measurement of both glucose oxidation (14CO2 production) and fatty acid oxidation (3H2O production). The contribution of fatty acid oxidation as a source of citric acid cycle acetyl-CoA was significantly greater in hearts from hibernating animals, compared to hearts from non-hibernating animals. Since acetyl-CoA carboxylase (ACC) regulates cardiac fatty acid oxidation (producing malonyl-CoA, a potent inhibitor of mitochondrial fatty acid uptake), we measured the activity and expression of ACC in these hearts. ACC activity was significantly decreased in hibernating ground squirrels, regardless of whether ACC was assayed at 37 degrees or 5 degrees C. This decrease in activity could not be explained by a change in the activity of 5'AMP-activated protein kinase, which can phosphorylate and inhibit ACC. Rather, the expression of the 280 kDa isoform of ACC (which predominates in cardiac muscle) was decreased in hearts from hibernating squirrel hearts. This suggests that a down regulation of ACC expression occurs as an adaptation for the increased utilization of fatty acid in hearts of hibernating ground squirrels.
The mechanism by which acyl-CoA dehydrogenases initiate catalysis was studied by using p-substituted phenylacetyl-CoAs (substituents-NO2, -CN, and CH3CO-), 3S-C8-, and 3'-dephospho-3S-C8CoA. These analogues lack a beta C-H and cannot undergo alpha,beta-dehydrogenation. Instead they deprotonate at alpha C-H at pH > or = 14 to form delocalized carbanions having strong absorbancies in the near UV-visible spectrum. The pKas of the corresponding phenylacetone analogues were determined as approximately 13.6 (-NO2), approximately 14.5 (-CN), and approximately 14.6 (CH3CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo alpha C-H deprotonation. While the extent of deprotonation varies, the anionic products from charge-transfer complexes with the oxidized flavin. From the pH dependence of the dissociation constants (Kd) of p-NO2-phenylacetyl-CoA (4NPA-CoA), 3S-C8-CoA, and 3'-dephospho-3S-C8CoA, four pKas at approximately 5, approximately 6, approximately 7.3, and approximately 8 were identified. They were assigned to the following ionizations: (a) pKa approximately 5, ligand (L-H) in the MCADH approximately ligand complex; (b) pKa approximately 6, Glu376-COOH in uncomplexed MCADH; (c) pKa approximately 7.3, Glu99-COOH in uncomplexed MCADH (Glu99 is a residue that flanks the bottom of the active-center cavity; this pK is absent in the mutant Glu99Gly-MCADH); and (d) pK approximately 8, Glu99-COOH in the MCADH approximately 4NPA-CoA complex. The pKa approximately 6 (b) is not significantly affected in the MCADH approximately 4NPA-CoA complex, but it is increased by > or = 1 pK unit in that with 3S-C8CoA and further in the presence of C8-CoA, the best substrate. The alpha C-H pKas of 4NPA-CoA, of 3S-C8-CoA, and of 3'-dephospho-3S-C8CoA in the complex with MCADH are approximately 5, approximately 5, and approximately 6. Compared to those of the free species these pKa values are therefore lowered by 8 to > or = 11 pH units (50 to > or = 65 kJ mol-1) and are close to the pKa of Glu376-COOH in the complex with substrate/ligand. This effect is ascribed mainly to the hydrogen-bond interactions of the thioester carbonyl group with the ribityl-2'-OH of FAD and Glu376-NH. It is concluded that the pKa shifts induced with normal substrates such as n-octanoyl-CoA are still higher and of the order of 9-13 pK units. With 4NPA-CoA and MCADH, alpha C-H abstraction is fast (kapp approximately 55 s-1 at pH 7.5 and 25 degrees C, deuterium isotope effect approximately 1.34). However, it does not proceed to completion since it constitutes an approach to equilibrium with a finite rate for reprotonation in the pH range 6-9.5. The extent of deprotonation and the respective rates are pH-dependent and reflect apparent pKas of approximately 5 and approximately 7.3, which correspond to those determined in static experiments.