Which Enzymes Are Impacted By Nadh Buildup In Glycolysis?

This article delves into the key aspects of glycolysis, including the formation of pyruvate, the yield of ATP, production of NADH, and the roles of enzymes throughout the process. Pyruvate formation is a central event in glycolysis, where NADH is generated when glyceraldehyde-3-phosphate is oxidized to 1,3-bisphosphoglycerate. Both NADH and its oxidized form, NAD+, play essential roles in various metabolic processes associated with cellular bioenergetics.

The “Pasteur effect” describes how the availability of oxygen diminishes the effect of glycolysis, and decreased availability leads to an acceleration of glycolysis initially. Under aerobic conditions, pyruvate can diffuse into mitochondria, where it enters the citric acid cycle and generates reducing equivalents in the form of NADH and FADH2.

Aerobic glycolysis is a series of reactions wherein oxygen is required to reoxidize NADH to NAD+, hence the name. This ten-step process begins with a molecule of glucose and ends up with two molecules of pyruvate. In the presence of ample oxygen, both pyruvate and NADH are transported into the mitochondria, where pyruvate enters the TCA cycle while NADH is oxidized to NAD+ by the electron transport chain. Mitochondrial NADH, gained from glycolysis or the TCA cycle, are oxidized by Complex I (NADH:ubiquinone oxidoreductase) of the ETC.

Excess NADH accumulation in conjunction with reduced NAD+ availability can inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH). NADH produced in glycolysis is utilized in reactions catalyzed by lactate dehydrogenase (LDH) or transported to the mitochondria for further processing.

The accumulation of NADH affects enzymes involved in glycolysis and pyruvate/lactate metabolism. The TIGAR protein, encoded by the C12orf5 gene, hinders the forward progression of glycolysis by creating a buildup of fructose. Water-forming NADH oxidase can oxidize cytosolic NADH to NAD+, thus relieving cytosolic NADH accumulation in Saccharomyces cerevisiae.

What happens to NADH at the end of glycolysis?

During aerobic glycolysis, this NADH is transported by the malate aspartate shuttle or glycerol phosphate shuttle to the mitochondria, where it is reoxidized to NAD+ while it participates in the electron transport chain to produce ATP.

Introduction. Glycolysis is a central metabolic pathway that is used by all cells for the oxidation of glucose to generate energy in the form of ATP (Adenosine triphosphate) and intermediates for use in other metabolic pathways. Besides glucose, other hexose sugars such as fructose and galactose also end up in the glycolytic pathway for catabolism.

Fundamentals. Glycolysis occurs in the cytoplasm where one 6 carbon molecule of glucose is oxidized to generate two 3 carbon molecules of pyruvate. The fate of pyruvate depends on the presence or absence of mitochondria and oxygen in the cells. The electron transport chain is the major site of oxygen consumption and the generation of ATP in the mitochondria. In cells with mitochondria, the pyruvate is decarboxylated by pyruvate dehydrogenase complex to form Acetyl-CoA that feeds into the Tricarboxylic acid cycle and ultimately participates in ATP production.

During the absence of oxygen (anaerobic conditions) and in the cells lacking mitochondria, anaerobic glycolysis prevails. The pyruvate is reduced to lactate as NADH is reoxidized to NAD+ by lactate dehydrogenase. This process is an important source of ATP for cells that lack mitochondria, such as erythrocytes. During aerobic glycolysis, this NADH is transported by the malate aspartate shuttle or glycerol phosphate shuttle to the mitochondria, where it is reoxidized to NAD+ while it participates in the electron transport chain to produce ATP.

Which enzymes are activated by the accumulation of citrate?

Citrate is a crucial component in cancer cells’ metabolism and regulation, derived from mitochondrial synthesis and/or carboxylation of α-ketoglutarate. It is cleaved by ATP-citrate lyase into acetyl-CoA and oxaloacetate, which are rapidly converted into acetyl-CoA and oxaloacetate, which sustain nucleotide synthesis and gluconeogenesis. In proliferative cancer cells, citrate levels are low, preventing its retro-inhibition on glycolytic enzymes. This regulation helps sustain the Warburg effect in cancer cells relying on glycolysis.

In those relying on an oxidative metabolism, fatty acid β-oxidation sustains a high production of citrate, which is still rapidly converted into acetyl-CoA and oxaloacetate, sustaining nucleotide synthesis and gluconeogenesis. Therefore, citrate levels are rarely high in cancer cells.

Citrate resistance to targeted therapies, such as tyrosine kinase inhibitors (TKIs), is often sustained by aerobic glycolysis and its key oncogenic drivers, such as Ras and its downstream effectors MAPK/ERK and PI3K/Akt. Preclinical cancer models have shown that high doses of citrate can inhibit glycolysis, promote cytotoxic drug sensibility and apoptosis, neutralize extracellular acidity, and inhibit tumor growth and key signaling pathways, including the IGF-1R/AKT pathway.

These preclinical results support the testing of citrate strategies in clinical trials to counteract key oncogenic drivers sustaining cancer development and resistance to anti-cancer therapies. Citrate is a gauge of nutrients available for biosynthesis and ATP production generated via oxidative phosphorylation (OXPHOS).

What happens if NADH builds up?

Major consequences of NADH accumulation include blockade of TCA turning, impairment of de novo aspartate synthesis, and resulting decreases in protein and nucleotide synthesis (Birsoy et al., 2015, Gameiro et al., 2013, Mullen et al., 2011, Sullivan et al., 2015, Wise et al., 2011, Yoo et al., 2008).

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Which enzyme of glycolysis is inhibited by the accumulation of citrate?

Recent research has shown that the addition of metabolic fuels like acetoacetate, fatty acids, lactate, or pyruvate to hearts perfused with glucose and insulin inhibits the rate of glucose uptake and oxidation. This inhibition is attributed to an indirect inhibition of phosphofructokinase, as indicated by increased levels of hexose monophosphates and decreased levels of fructose-1, 6-diphosphate. Furthermore, phosphofructokinase activity has been depressed in hearts from diabetic rats, which also have a decreased rate of glucose utilization. Citrate not only inhibits phosphofructokinase activity in cell-free systems but also accumulates in hearts from diabetic rats and isolated hearts perfused with long- and short-chain fatty acids. This suggests that the rate of glycolysis may be controlled by the activity of the citric acid cycle. Williamson reported that the rate-limiting step of glycolysis in perfused hearts with pyruvate occurred at a site located between glyceraldehyde-3-phosphate and pyruvate. The study aims to examine the kinetics of changes of glycolytic intermediates after the addition of pyruvate, in conjunction with the levels of certain citric acid cycle intermediates in both the intact heart and isolated heart mitochondria.

What enzymes does NADH inhibit?

The study focuses on the inhibition of the αγ, αβ, and α2βγ enzymes of human NAD-IDH. Previous biochemical studies have shown that the enzymatic activity of NAD-IDH purified from bovine heart can be inhibited by NADH. Isocitrate dehydrogenases (IDHs) are enzymes that catalyze the oxidative decarboxylation of isocitrate into α-ketoglutarate (α-KG) and CO 2 while converting the coenzyme NAD+ or NADP + into NADH or NADPH. In mammals, the mitochondria localized NAD-dependent IDHs (NAD-IDHs) are deemed to exert the catalytic role in the citric acid cycle.

Mammalian NAD-IDH functions as a heterotetramer consisting of two α subunits (37 kDa), one β subunit (39 kDa), and one γ subunit (39 kDa). The α and β subunits form a heterodimer (αβ), while the α and γ subunits form another (αγ), which can be further assembled into a heterooctamer.

Previous biochemical studies have shown that the enzymatic activity of mammalian NAD-IDH could be positively regulated by citrate (CIT) and ADP. In the α 2 βγ heterotetramer, the α subunit exerts the catalytic role, while the β and γ subunits play the regulatory roles. However, the molecular basis for the assembly of the α 2 βγ heterotetramer and the molecular mechanisms of the allosteric regulation of the α 2 βγ heterotetramer are still elusive due to the lack of structural information about the α 2 βγ heterotetramer.

The study also examined the inhibition of the αβ and αγ heterodimers and the α 2 βγ heterotetramer of human NAD-IDH by NADH, finding that all these enzymes can be substantially inhibited by NADH. The crystal structure of the αγ heterodimer bound with an Mg 2+ and an NADH at the active site and an NADH at the allosteric site was solved to understand the molecular mechanism of the NADH inhibition.

The kinetic data confirmed that the NADH binding competes with the binding of NAD+ to the active site and the binding of CIT and ADP to the allosteric site, contributing to the inhibition of NADH on the αγ heterodimer. These findings provide insights into the inhibitory mechanism of the αγ heterodimer by NADH.

How does NADH affect glycolysis?

NAD+ is a crucial metabolite in the human body that plays a vital role in cellular energy metabolism, glycolysis, and redox reactions. It was first described in 1906 as a component that could increase fermentation rate in yeast and later became a vital redox carrier. NAD+ receives hydride from metabolic processes like glycolysis, the TCA cycle, and fatty acid oxidation to form NADH, which serves as a central hydride donor to ATP synthesis through mitochondrial OXPHOS and generates reactive oxygen species (ROS). NAD+ also serves as a co-substrate for various enzymes, including sirtuins, PARPs, CD157, CD73, CD38, and SARM.

In response to cellular stress and physiological stimuli, NAD+ and its metabolites levels rewire biological processes via post-synthesis modification of fundamental biomolecules, including DNA, RNA, and proteins. These activities impact energy metabolism, DNA repair, epigenetic modification, inflammation, circadian rhythm, and stress resistance. NAD+ deficiency contributes to a spectrum of diseases including metabolic diseases, cancer, aging, and neurodegeneration disorders. Recent advances in understanding NAD+ homeostasis in response to growth conditions or environmental stimuli highlight its actions in coordinating metabolic reprogramming and maintaining cellular physiologic biology. NAD+ and its metabolites serve as essential hubs in both physiological and pathophysiological processes and have the potential to be modulated in the clinical treatment of diseases.

What happens to the NADH produced in glycolysis in the presence of oxygen?

The NADH that is produced in glycolysis may either enter the mitochondria and donate is electrons to the transport chain, or may be used in fermentative pathways. This depends on the presence of oxygen. If there is oxygen available, NADH will enter the mitochondria; if not it will enter fermentation.

Which of the following enzymes would be inhibited by the addition of NADH?

All enzymes that have NADH as a product would be inhibited by the addition of NADH. The correct answers are citrate synthase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase. Pyruvate dehydrogenase, which synthesizes acetyl-CoA from pyruvate, is also inhibited by NADH.

Glucose is converted to pyruvate through glycolysis, and pyruvate must then be converted into acetyl-CoA in order to enter the citric acid cycle.

What is the name of the enzyme that catalyzes the conversion of pyruvate into acetyl-CoA?

What enzymes inhibit glycolysis?

Most of the reported glycolysis inhibitors are summarized (Table 1 and Figure 1). The enzymes targeted include hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), and pyruvate dehydrogenase kinase (PDK).

What happens if there’s too much NADH?

Redox imbalance in diabetes can have detrimental effects, as excess NADH accumulates, inhibiting enzymes that produce NADH from NAD+, leading to potential reactive oxygen species (ROS) production. This can also overload mitochondrial electron transport chains, causing oxidant overload on complex I, a major site for ROS generation. The more NADH it oxidizes, the more ROS it produces, leading to oxidative damage to proteins, DNA, and lipids. These oxidized macromolecules can accumulate over time, manifesting diabetic glucotoxicity, insulin resistance, β-cell insulin deficiency, global cell death, and tissue dysfunction. Oxidative damage and oxidative stress have been demonstrated to be involved in the pathogenesis of diabetes and its complications. Inhibition of complex I has been shown to activate 5′-AMP-activated protein kinase and improve glucose metabolism in diabetes. Restoring redox balance or attenuating oxidative stress could be a promising approach to treating these chronic age-related diseases. Additionally, the roles of antioxidants in antidiabetic therapy should be appreciated, as antioxidants usually work by ultimately improving redox balance. Redox imbalance can elevate cellular levels of ROS that can attack proteins, DNA, and lipids, causing cell death and tissue dysfunction, which is thought to be involved in the pathogenesis of diabetes and its complications.

Which enzymes will be inhibited if excessive NADH begins to accumulate?

NADH, a key regulatory enzyme in the TCA cycle, inhibits all regulatory enzymes, leading to the shut-down of the cycle when ETC malfunctions. ATP, generated through the ETC and OXPHOS, is also an allosteric inhibitor of pyruvate dehydrogenase (PDH) and IDH. Acetyl-CoA, a thioester between two-carbon acetyl groups and a thiol, coenzyme A (CoA), is crucial for maintaining the TCA cycle activity. It can be generated from various sources and compartments, including mitochondria, cytosol, and cancer cells.

Acetyl-CoA regulates chromatin dynamics by providing acetyl groups for acetylation, a major post-translational protein modification in the cell. It plays a crucial role in the acetylation of histones, which alters chromatin dynamics to drive epigenetic control of gene expression by activating transcriptional programs. Histone acetyltransferases (HATs) catalyze the addition of acetyl groups in histone N-terminal tails, and changes in acetyl-CoA levels affect global histone acetylation and gene expression.

The TCA cycle’s activity is essential to provide metabolites that control chromatin modifications and DNA methylation. Histone acetylation by HATs is dependent on the availability of acetyl-CoA, which provides the necessary acetyl groups to enable the reaction. Acetyl-CoA is produced in the cytosol by ACLY using citrate exported from the TCA cycle in mitochondria.

α-ketoglutarate (α-KG) is an essential cofactor of 2-OGDD, including histone demethylases JMJDs and TET DNA demethylases. Succinate, the product of 2-OGDD enzyme reactions, works as an antagonist of the reaction when it accumulates. 2-HG and fumarate can also rewire the epigenetic landscape of cells through inhibition of histone and DNA demethylases.