GLYCOLYSIS CONCERNS

GLYCOLYSIS CONCERNS  Although glycolysis is a nearly universal process, the fate of its end product, pyruvate, may vary in different organisms or even in different tissues.  In the presence of oxygen, the most common situation in multicellular organisms and many unicellular ones, pyruvate is metabolized to carbon dioxide and water through the citric acid cycle and the electron transport chain.  In the absence of oxygen, fermentation generates a lesser amount of energy; pyruvate is converted, or fermented, into lactic acid in lactic acid fermentation or into ethanol in alcoholic fermentation.  Lactic acid production takes place in skeletal muscle when energy needs outpace the ability to transport oxygen.  Glycolysis is a catabolic pathway in the cytoplasm that is found in almost all organisms—irrespective of whether they live aerobically or anaerobically.  In eukaryotic cells, glycolysis takes place in the cytosol. This pathway can be thought of as comprising three stages  Stage 1, which is the conversion of glucose into fructose 1, 6bisphosphate, consists of three steps: a phosphorylation, an isomerization, and a second phosphorylation reaction.

 The strategy of these initial steps in glycolysis is to trap the glucose in the cell and form a compound that can be readily cleaved into phosphorylated three-carbon units.  Stage 2 is the cleavage of the fructose 1,6-bisphosphate into two three-carbon fragments. These resulting three-carbon units are readily interconvertible.  In stage 3, ATP is harvested when the three carbon fragments are oxidized to pyruvate.  Glycolysis involves ten individual steps, including three isomerizations and four phosphate transfers. The only redox reaction takes place in step [6].  [1] Glucose, which is taken up by animal cells from the blood and other sources, is first phosphorylated to glucose 6phosphate, with ATP being consumed. The glucose 6-phosphate is not capable of leaving the cell.  [2] In the next step, glucose 6-phosphate is isomerized into fructose 6-phosphate.  [3] Using ATP again, another phosphorylation takes place, giving rise to fructose 1,6-bisphosphate. Phosphofructokinase is the most important key enzyme in glycolysis.

 [4] Fructose 1,6-bisphosphate is broken down by aldolase into the C3 compounds glyceraldehyde3-phosphate (also known as glyceral3-phosphate) and glycerone3-phosphate (dihydroxyacetone 3-phosphate).  [5] The latter two products are placed in fast equilibrium by triosephosphate isomerase.  [6] Glyceraldehyde 3-phosphate is now oxidized by +

glyceraldehyde-3-phosphate dehydrogenase, with NADH + H

being formed. In this reaction, inorganic phosphate is taken up into the molecule (substrate-level phosphorylation; and 1,3bisphosphoglycerate is produced. This intermediate contains a mixed acid–anhydride bond, the phosphate part of which is at a high chemical potential.  [7] Catalyzed by phosphoglycerate kinase, this phosphate residue is transferred to ADP, producing 3-phosphoglycerate and ATP. The ATP balance is thus once again in equilibrium.  [8] As a result of shifting of the remaining phosphate residue within the molecule, the isomer 2-phosphoglycerate is formed.  [10] In the last step, pyruvate kinase transfers this residue to ADP. The remaining enol pyruvate is immediately rearranged into pyruvate, which is much more stable.

SOME HIGHLIGHTS  *1*Glucose is phosphorylated by ATP to form glucose 6phosphate. This step is notable for two reasons: (1) glucose 6phosphate cannot diffuse through the membrane, because of its negative charges, and (2) the addition of the phosphoryl group begins to destabilize glucose, thus facilitating its further metabolism.

 The glucose-induced structural changes are significant in two respects. First, the environment around the glucose becomes much more nonpolar, which favors the donation of the terminal phosphoryl group of ATP. Second, the substrate-induced conformational changes within the kinase enable it to discriminate against H2O as a substrate. That means it blocks the access of water (from the solvent), which might otherwise enter the active site and attack (hydrolyze) the phosphoanhydride bonds (esp., the γ phosphoryl group) of ATP forming ADP and Pi. In other words, a rigid kinase would necessarily also be an ATPase.

 Like the other nine enzymes of glycolysis, hexokinase is a soluble, cytosolic protein.  Hexokinase, like adenylate kinase and all other kinases, requires Mg 2 + (or another divalent metal ion such as Mn2+) for activity. The divalent metal ion forms a complex with ATP.

 Kinetic studies of NMP kinases, as well as many other enzymes having ATP or other nucleoside triphosphates as a substrate, reveal that these enzymes are essentially inactive in the absence of divalent metal ions such as magnesium (Mg2+) or manganese (Mn2+), but acquire activity on the addition of these ions.  The metal is not a component of the active site. Rather, nucleotides such as ATP bind these ions, and it is the metal ionnucleotide complex that is the true substrate for the enzymes.  Essentially all nucleoside triphosphates are present as NTPMg2+ complexes.  (1) The magnesium ion neutralizes some of the negative charges present on the polyphosphate chain, reducing nonspecific ionic interactions between the enzyme and the polyphosphate group of the nucleotide.

 (2) The interactions between the magnesium ion and the oxygen atoms in the phosphoryl group hold the nucleotide in welldefined conformations that can be specifically bound by the enzyme.  (3) The magnesium ion provides additional points of interaction between the ATP-Mg2+ complex and the enzyme, thus increasing the binding energy.

 **In some cases, such as the DNA polymerases, side chains (often aspartate and glutamate residues) of the enzyme can bind directly to the magnesium ion. In other cases, the enzyme interacts indirectly with the magnesium ion through hydrogen bonds to the coordinated water molecules (Figure 9.50). Such interactions have been observed in adenylate kinases bound to ATP analogs.  Hexokinase is present in all cells of all organisms. Hepatocytes also contain a form of hexokinase called hexokinase IV or glucokinase, which differs from other forms of hexokinase in kinetic and regulatory properties.  Two enzymes that catalyze the same reaction but are encoded in different genes are called isozymes.  *2* The enzyme phosphohexose isomerase (phosphoglucose isomerase) catalyzes the reversible isomerization of glucose 6phosphate, an aldose, to fructose 6-phosphate, a ketose.

 The enzyme must first open the six-membered ring of glucose 6phosphate, catalyze the isomerization, and then promote the formation of the five-membered ring of fructose 6-phosphate.

 *3* Phosphorylation of Fructose 6-Phosphate to Fructose 1,6Bisphosphate, phosphofructokinase-1 (PFK-1) catalyzes the transfer of a phosphoryl group from ATP to fructose 6phosphate to yield fructose 1,6-bisphosphate.

 This enzyme is called PFK-1 to distinguish it from a second enzyme (PFK-2) that catalyzes the formation of fructose 2,6bisphosphate from fructose 6-phosphate in a separate pathway.  The PFK-1 reaction is essentially irreversible under cellular conditions.  Phosphofructokinase-1 is a regulatory enzyme, one of the most complex known.  PFK1 is the major point of regulation in glycolysis. The activity of PFK-1 is increased whenever the cell’s ATP supply is depleted or when the ATP breakdown products, ADP and AMP (particularly the latter), are in excess. The enzyme is inhibited whenever the cell has ample ATP and is well supplied by other fuels such as

fatty acids. In some organisms, fructose 2,6-bisphosphate is a potent allosteric activator of PFK-1.  The enzyme fructose 1,6-bisphosphate aldolase, often called simply aldolase, catalyzes a reversible aldol condensation.  *4* Fructose 1,6-bisphosphate is cleaved to yield two different triose phosphates, glyceraldehyde 3-phosphate (GAP) , an aldose, and dihydroxyacetone phosphate (DHAP) , a ketose.

 Although the aldolase reaction has a strongly positive standard free-energy change in the direction of fructose 1,6-bisphosphate cleavage, at the lower concentrations of reactants present in cells, the actual free-energy change is small and the aldolase reaction is readily reversible.

 Only one of the two triose phosphates formed by aldolase, glyceraldehyde 3-phosphate, can be directly degraded in the subsequent steps of glycolysis.  *5* The other product, dihydroxyacetone phosphate, is rapidly and reversibly converted to glyceraldehyde 3-phosphate by the fifth enzyme of the sequence, triose phosphate isomerase.

 This reaction is rapid and reversible. At equilibrium, 96% of the triose phosphate is dihydroxyacetone phosphate.  *5* TIM catalyzes the transfer of a hydrogen atom from carbon 1 to carbon 2 in converting dihydroxyacetone phosphate into glyceraldehyde 3-phosphate, an intramolecular oxidationreduction.

 Two features of this enzyme are noteworthy. First, TIM displays great catalytic prowess. Indeed, the k cat/K M ratio for isomerization of glyceraldehyde 3-phosphate is 2 × 108 M-1 s-1, which is close to the diffusion-controlled limit. In other words, the rate-limiting step in catalysis is the diffusion-controlled encounter of substrate and enzyme. TIM is an example of a kinetically perfect enzyme. Second, TIM suppresses an undesired side reaction, the decomposition of the enediol intermediate into methyl glyoxal and inorganic phosphate.  The payoff phase of glycolysis includes the energy-conserving phosphorylation steps in which some of the free energy of the glucose molecule is conserved in the form of ATP.  One molecule of glucose yields two molecules of glyceraldehyde 3-phosphate; both halves of the glucose molecule follow the same pathway in the payoff phase of glycolysis.

 *6* The first step in the payoff phase is the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, catalyzed by glyceraldehyde 3-phosphate dehydrogenase.

 This is the first of the two energy-conserving reactions of glycolysis that eventually lead to the formation of ATP.  The aldehyde group of glyceraldehyde 3-phosphate is oxidized, not to a free carboxyl group but to a carboxylic acid anhydride with phosphoric acid.  This type of anhydride, called an acyl phosphate, has a very high standard free energy of hydrolysis. So 1,3-Bisphosphoglycerate is an acyl phosphate.

 Such compounds have a high phosphoryl-transfer potential; one of its phosphoryl groups is transferred to ADP in the next step in glycolysis.  The reaction is the sum of two processes: the oxidation of the aldehyde to a carboxylic acid by NAD+ and the joining of the carboxylic acid and orthophosphate to form the acyl-phosphate product.  Much of the free energy of oxidation of the aldehyde group of glyceraldehyde 3-phosphate is conserved by formation of the acyl phosphate group at C-1 of 1,3-bisphosphoglycerate.  The acceptor of hydrogen in the glyceraldehyde 3-phosphate dehydrogenase reaction is NAD+ , bound to a Rossmann fold. The reduction of NAD+ proceeds by the enzymatic transfer of a hydride ion (:H-) from the aldehyde group of glyceraldehyde 3phosphate to the nicotinamide ring of NAD+, yielding the reduced coenzyme NADH. The other hydrogen atom of the substrate molecule is released to the solution as H+ .  Glyceraldehyde 3-phosphate is covalently bound to the dehydrogenase during the reaction. The aldehyde group of glyceraldehyde 3-phosphate reacts with the --SH group of an essential Cys residue in the active site, in a reaction analogous to the formation of a hemiacetal, in this case producing a thiohemiacetal. Reaction of the essential Cys residue with a heavy metal such as Hg2+ irreversibly inhibits the enzyme.

 IN OTHER WORDS  Let us consider the mechanism of glyceraldehyde 3-phosphate dehydrogenase in detail.  In step 1, the aldehyde substrate reacts with the sulfhydryl group of cysteine 149 on the enzyme to form a hemithioacetal.  Step 2 is the transfer of a hydride ion to a molecule of NAD + that is tightly bound to the enzyme and is adjacent to the cysteine residue. This reaction is favored by the deprotonation of the hemithioacetal by histidine 176. The products of this reaction are the reduced coenzyme NADH and a thioester intermediate. This thioester intermediate has a free energy close to that of the reactants.  In step 3, orthophosphate attacks the thioester to form 1,3-BPG and free the cysteine residue. This displacement occurs only after the NADH formed from the aldehyde oxidation has left the enzyme and been replaced by a second NAD+. The positive charge on the NAD+ may help polarize the thioester intermediate to facilitate the attack by orthophosphate.  The favorable oxidation and unfavorable phosphorylation reactions are coupled by the thioester intermediate, which preserves much of the free energy released in the oxidation reaction.

 *7* The enzyme phosphoglycerate kinase transfers the high-energy phosphoryl group from the carboxyl group of 1,3bisphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate. 

 Notice that phosphoglycerate kinase is named for the reverse reaction. Like all enzymes, it catalyzes the reaction in both directions. This enzyme acts in the direction suggested by its name during gluconeogenesis and during photosynthetic CO2 assimilation.  This Substrate-level phosphorylation (SLP) involves soluble enzymes and chemical intermediates (1,3-bisphosphoglycerate in this case).  The formation of ATP in this manner is referred to as substratelevel phosphorylation because the phosphate donor, 1,3-BPG, is a substrate with high phosphoryl-transfer potential.  Thus, the outcomes of the reactions catalyzed by glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase are: 1. Glyceraldehyde 3-phosphate, an aldehyde, is oxidized to 3phosphoglycerate, a carboxylic acid. 2. NAD+ is concomitantly reduced to NADH. 3. ATP is formed from Pi and ADP at the expense of carbon oxidation energy. *8* The enzyme phosphoglycerate mutase catalyzes a reversible shift of the phosphoryl group between C-2 and C-3 of glycerate; Mg2+ is essential for this reaction.

 Bisphosphoglycerate mutase catalyzes the conversion of 1,3bisphosphoglycerate to 2,3-bisphosphoglycerate, which is converted to 3-phosphoglycerate by 2,3-bisphosphoglycerate phosphatase (and possibly also phosphoglycerate mutase). This alternative pathway involves no net yield of ATP from glycolysis. However, it does serve to provide 2,3-bisphosphoglycerate, which binds to hemoglobin, decreasing its affinity for oxygen and so making oxygen more readily available to tissues.  In general, a mutase is an enzyme that catalyzes the intramolecular shift of a chemical group, such as a phosphoryl group.  This enzyme requires catalytic amounts of 2,3-bisphosphoglycerate to maintain an active-site histidine residue in a phosphorylated form.

 Examination of the first partial reaction reveals that the mutase may function as a phosphatase it converts 2,3-bisphosphoglycerate into 2-phosphoglycerate. However, the phosphoryl group remains linked to the enzyme. This phosphoryl group is then transferred to 3-phosphoglycerate to reform 2,3-bisphosphoglycerate.

 *9* In the second glycolytic reaction that generates a compound with high phosphoryl group transfer potential, enolase promotes reversible removal of a molecule of water (dehydration) from 2-phosphoglycerate to yield phosphoenolpyruvate (PEP).  Although 2-phosphoglycerate and phosphoenolpyruvate contain nearly the same total amount of energy, the loss of the water molecule from 2-phosphoglycerate causes a redistribution of energy within the molecule, greatly increasing the standard free energy of hydrolysis of the phosphoryl group.

 *10* The last step in glycolysis is the transfer of the phosphoryl group from phosphoenolpyruvate to ADP, catalyzed by pyruvate kinase, which requires K+ and either Mg2+ or Mn2+ .

 In this substrate-level phosphorylation, the product pyruvate first appears in its enol form, then tautomerizes rapidly and nonenzymatically to its keto form, which predominates at pH 7.

 The overall reaction has a large, negative standard free energy change, due in large part to the spontaneous conversion of the enol form of pyruvate to the keto form.  The pyruvate kinase reaction is essentially irreversible under intracellular conditions and is an important site of regulation.  Note that the energy released in the anaerobic conversion of glucose into two molecules of pyruvate is -21 kcal mol-1 (-88 kJ mol-1).

 In aerobic respiration, Electron transfer from NADH to O2 in mitochondria provides the energy for synthesis of ATP by respiration linked phosphorylation.  Glycolysis is tightly regulated in coordination with other energyyielding pathways to assure a steady supply of ATP. Hexokinase, PFK-1, and pyruvate kinase are all subject to allosteric regulation that controls the flow of carbon through the pathway and maintains constant levels of metabolic intermediates.

 The flux of glucose through the glycolytic pathway is regulated to maintain nearly constant ATP levels (as well as adequate supplies of glycolytic intermediates that serve biosynthetic roles).  The required adjustment in the rate of glycolysis is achieved by a complex interplay among ATP consumption, NADH regeneration, and allosteric regulation of several glycolytic enzymes—including hexokinase, PFK-1, and pyruvate kinase— and by second-to-second fluctuations in the concentration of key metabolites that reflect the cellular balance between ATP production and consumption.  On a slightly longer time scale, glycolysis is regulated by the hormones glucagon, epinephrine, and insulin, and by changes in the expression of the genes for several glycolytic enzymes.

…MEDICAL CORRELATIONS…  Glucose uptake and glycolysis proceed about ten times faster in most solid tumors than in noncancerous tissues. Tumor cells commonly experience hypoxia (limited oxygen supply), because they initially lack an extensive capillary network to supply the tumor with oxygen. As a result, cancer cells more than 100 to 200 m from the nearest capillaries depend on anaerobic glycolysis for much of their ATP production. They take up more glucose than normal cells, converting it to pyruvate and then to lactate as they recycle NADH.  The hypoxia-inducible transcription factor (HIF-1) is a protein that acts at the level of mRNA synthesis to stimulate the synthesis of at least eight of the glycolytic enzymes. This gives the tumor cell the capacity to survive anaerobic conditions until the supply of blood vessels has caught up with tumor growth.  2,3-bisphosphoglycerate binds to hemoglobin, decreasing its affinity for oxygen and so making oxygen more readily available to tissues.  Arsenite and mercuric ions react with the --SH groups of lipoic acid and inhibit pyruvate dehydrogenase, as does a dietary deficiency of thiamin, allowing pyruvate to accumulate.  Nutritionally deprived alcoholics are thiamin-deficient and may develop potentially fatal pyruvic and lactic acidosis.

 Patients with inherited pyruvate dehydrogenase deficiency, which can be due to defects in one or more of the components of the enzyme complex, also present with lactic acidosis, particularly after a glucose load. Because of its dependence on glucose as a fuel, brain is a prominent tissue where these metabolic defects manifest themselves in neurologic disturbances.  Deficiency of pyruvate kinase causes decreased production of ATP from glycolysis. Red blood cells have insufficient ATP for their membrane pumps, and a hemolytic anemia results, although oxygen delivery to tissues is not necessarily affected. As phosphoenolpyruvate accumulates, it is converted to 2phosphoglycerate, which leads to increased levels of 2,3bisphosphoglycerate in the red blood cells. The elevated levels of 2,3-bisphosphoglycerate promote oxygen release from hemoglobin in the tissues to an extent that is greater than in the presence of normal 2,3-bisphosphoglycerate levels.  Inherited aldolase A deficiency and pyruvate kinase deficiency in erythrocytes cause hemolytic anemia. The exercise capacity of patients with muscle phosphofructokinase deficiency is low, particularly on high-carbohydrate diets. By providing an alternative lipid fuel, e.g., during starvation, when blood free fatty acids and ketone bodies are increased, work capacity is improved.

 GLUT1 deficiency can have serious consequences. The GLUT1 transporter translocates glucose across the blood–brain barrier. When one allele is defective, the rate of glucose entry into the nervous system is insufficient for the cells’ needs, leading to seizures, developmental delays, and microcephaly. The treatment consists of a ketogenic diet, one high in fat, in order to produce ketone bodies as an alternative energy source for the nervous system.  An increase of lactate levels in the blood causes an acidosis (lactic acidosis). This condition can result from hypoxia or alcohol ingestion. Lack of oxygen slows down the electron transport chain, resulting in increased NADH levels. High NADH levels cause more than normal amounts of pyruvate to be converted to lactate. High NADH levels from alcohol metabolism also cause increased conversion of pyruvate to lactate. Thiamine deficiency, which is common in alcoholics, decreases pyruvate dehydrogenase activity, causing pyruvate to accumulate and form lactate. Thiamine deficiency also slows down the TCA cycle at the α-ketoglutarate dehydrogenase step. This and other conditions that slow down the TCA cycle can also produce a lactic acidosis.

Credit goes to----.  Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme Usage subject to terms and conditions of license. All right preserved.

 Harper’s Illustrated Biochemistry, Twenty-Sixth Edition  LUBERT STRYER Biochemistry, Fifth Edition  Lehninger principles of biochemistry 4th ED.  BRS Biochemistry, Molecular Biology, and Genetics 6TH ED.

SUMMARIZED BY// ALY BARAKAT