Abstract
The enzyme phosphofructokinase-1 (PFK-1) catalyzes the first committed step of glycolysis and is regulated by a complex array of allosteric effectors that integrate glycolytic flux with cellular bioenergetics. Here, we demonstrate the direct, potent, and reversible inhibition of purified rabbit muscle PFK-1 by low micromolar concentrations of long chain fatty acyl-CoAs (apparent Ki∼1 μM). In sharp contrast, short chain acyl-CoAs, palmitoylcarnitine, and palmitic acid in the presence of CoASH were without effect. Remarkably, MgAMP and MgADP but not MgATP protected PFK-1 against inhibition by palmitoyl-CoA indicating that acyl-CoAs regulate PFK-1 activity in concert with cellular high energy phosphate status. Furthermore, incubation of PFK-1 with [1-(14)C]palmitoyl-CoA resulted in robust acylation of the enzyme that was reversible by incubation with acyl-protein thioesterase-1 (APT1). Importantly, APT1 reversed palmitoyl-CoA-mediated inhibition of PFK-1 activity. Mass spectrometric analyses of palmitoylated PFK-1 revealed four sites of acylation, including Cys-114, Cys-170, Cys-351, and Cys-577. PFK-1 in both skeletal muscle extracts and in purified form was inhibited by S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, demonstrating that covalent acylation of PFK-1 was not required for inhibition. Tryptic footprinting suggested that S-hexadecyl-CoA induced a conformational change in PFK-1. Both palmitoyl-CoA and S-hexadecyl-CoA increased the association of PFK-1 with Ca2+/calmodulin, which attenuated the binding of palmitoylated PFK-1 to membrane vesicles. Collectively, these results demonstrate that fatty acyl-CoA modulates phosphofructokinase activity through both covalent and noncovalent interactions to regulate glycolytic flux and enzyme membrane localization via the branch point metabolic node that mediates lipid flux through anabolic and catabolic pathways.
Highlights
Glycolysis occupies a central role in eukaryotic energy metabolism through the production of NADH and ATP to meet immediate cellular energy demands, the generation of glycerol 3-phosphate for the de novo synthesis of phospholipids and triglycerides, and the production of pyruvate that is converted to acetyl-CoA for subsequent utilization in the TCA cycle or for de novo fatty acid biosynthesis
The ability of fatty acids to suppress glycolytic flux has been known for 50 years [1,2,3,4], the mechanisms by which metabolically active tissues undergo a rapid shift from glucose utilization to fatty acid oxidation during metabolic transitions remain incompletely understood
Because mammalian PFKs are known to contain an allosteric activation site and a catalytic site, we considered the possibility that acylCoAs could bind to an adenine-based regulatory site and modulate PFK-1 activity to integrate glycolytic flux with fatty acid oxidation
Summary
We identified a direct mechanism through which the branch point metabolite in fatty acid anabolic and catabolic metabolism, acyl-CoA, regulates the rate-determining and first committed step of glycolysis, the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by PFK-1. Both naturally occurring long chain fatty acyl-CoAs as well as a synthetic nonhydrolyzable analog (S-hexadecyl-CoA) resulted in potent inhibition of PFK-1 that was blocked by low concentrations of AMP and ADP (200 M) but not ATP. These results identify multiple mechanisms by which fatty acyl-CoAs promote alterations in PFK activity, PFK-membrane interactions, and PFK-regulatory protein associations that allow physiologic metabolic responses during health and likely compromise physiologic metabolic substrate transitions in lipid-related disease states in which fatty acylCoAs accumulate
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